Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20)) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey(R)) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required

Temporal and pharmacological characterization of angiostatin release and generation by human platelets: implications for endothelial cell migration.

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation

Cell density optimization for the in-cell western assay.

<p>Uterine myocytes were seeded at densities of 500 (darkest histogram in each grouping), 550, 600 and 650 (lightest histogram in each grouping) cells/mm². Cells were treated with increasing concentrations of OT (10⁻¹⁰ to 10⁻⁷ M) for 20, 30, 45 or 60 seconds or 2 or 5 minutes (n = 4 at each time point at each concentration of OT). Concentrations of PMLC₂₀ were measured using the ICW assay. PMLC₂₀ levels rose rapidly (20 sec), decayed significantly by 1 min, and returned to baseline levels by 5 min. The largest amplitude concentration-dependent change in PMLC₂₀ levels was measured at 20 sec and 600 cells/mm².</p

Signal linearity with the in-cell western assay.

<p><b>A.</b> 96-well microplates were loaded with an increasing number of cells/well. Signals from anti-GAPDH and anti-PMLC₂₀ antibodies appear as green fluorophores. Signals from cell dyes (cell number normalization) appear as red fluorophores. <b>B.</b> Quantification of signals shown in panel A. Normalized values are expressed as a fraction of the signal intensity of the wells containing 15,000 cells for GAPDH (•), PMLC₂₀ (▴). The signal from the cell dyes also is shown (▪). The GAPDH and PMLC₂₀ signals appear to plateau at the higher cell densities so the line of best fit has been calculated and illustrated by expressing the normalized values as a fraction of the intensity of the wells containing 10,500 cells (inset) for GAPDH (•), and PMLC₂₀ (▴).</p

Antibody specificity using the in-cell western assay.

<p>In each pair of micrographs, the left panel illustrates filamentous actin (F-actin) stained with rhodamine-phalloidin (red). The corresponding right panels are immunofluorescence micrographs stained with antibodies conjugated to Alexa-Fluor 488 (green). Nuclei are stained with DAPI (blue) in all panels. Images are shown at 400× and 200× magnification in panels A and B, respectively. White bars represent 25 and 50 microns, respectively. <b>A.</b> Demonstration of GAPDH and total MLC₂₀. Panels i and ii. The actin fibers stain in a filamentous pattern typical of uterine smooth muscle. There is no detectable signal with omission of the primary antibodies. Panels iii and iv. The GAPDH staining shows a diffuse cytosolic pattern in contrast to the fibrillar pattern of actin. Panels v and vi. Total MLC has a similar staining pattern to actin. <b>B.</b> Demonstration of PMLC₂₀. Panels i–iv. There is no detectable background fluorescence when the antibody has been preadsorbed with blocking peptide (BP) containing phospho-Ser¹⁹ of MLC₂₀, either in the resting state (panel ii) or with stimulation using 100 nM OT (20 sec stimulus, panel iv). Only a small amount of PMLC₂₀ is detectable in the resting myocyte (panel vi) but this is markedly increased upon stimulation with OT (100 nM, 20 sec: panel viii).</p

Localization of angiostatin to platelet α-granules.

<p>(<b>A</b>) Represenative platelet immunogold-electron microscopy and (<b>B</b>) confocal immunofluorescence microscopy. N = 3.</p

Effect of anti-platelet agents on angiostatin release.

<p>(<b>A</b>) Represenative traces and (<b>B</b>) summary data demonstrating the inhibitory effects of ASA (100 µM) and RGDS (10 µM) on platelet aggregation. (<b>C</b>) Represenative immunoblot and summary densitometry data demonstrating the inhibitory effects of ASA (100 µM) and RGDS (10 µM) on platelet angiostatin release. (<b>D</b>) Summary data demonstrating the inhibitory effects of ASA (100 µM) and RGDS (10 µM) on platelet VEGF release. (<b>E</b>) Represenative traces demonstrating the inhibitory effects of MRS2395 (50 µM) on platelet aggregation. (<b>F</b>) Represenative immunoblot and summary densitometry data demonstrating that MRS2395 does not inhibit angiostatin release. N = 4. *, P<0.05 vs. control. Angst – Angiostatin.</p

Effect of physiological platelet inhibitors on angiostatin release.

<p>(<b>A</b>) Summary data demonstrating the concentration inhibitory effects of PGI₂ on platelet aggregation. (<b>B</b>) Representative immunoblot and summary densitometry data demonstrating the inhibitory effects of PGI₂ on platelet angiostatin release. (<b>C</b>) Summary data demonstrating the concentration inhibitory effects of the nitric oxide donor GSNO on platelet aggregation. (<b>D</b>) Representative immunoblot and summary densitometry data demonstrating the inhibitory effects of GSNO on platelet angiostatin release. Collagen (3 µg/ml) was used to induce aggregation. N = 5. *, P<0.05 vs. control. Angst – Angiostatin.</p

Suppression of platelet angiostatin generation enhances endothelial cell migration in the presence of ASA.

<p>Representative photomicrographs and summary data demonstrating the inhibitory effects of ASA (100 µM) on platelet-stimulated HMVEC-C migration under hypoxic conditions. Inhibition of platelet angiostatin generation by aprotinin (30 µM) reverses the migration inhibitory effects of ASA-inhibited platelet releasates. Platelets were aggregated by collagen (3 µg/ml). ASA was added to platelet rich plasma during platelet preparation and then subsequently washed away. An equal concentration of aprotinin was added to control and ASA-treated platelet releasates following aggregation. This was done to account for any potential affects of aprotinin in the releasates on the migration assays. Scale bars = 100 µm. N = 4. *, P<0.05 vs. control.</p

Angiostatin suppresses platelet-stimulated endothelial cell migration.

<p>Representative HMVEC-C migration photomicrographs and summary data. HMVEC-C migration in response to platelet releasates from collagen (3 µg/ml) activated (during platelet shape change) and maximum aggregated platelets under hypoxic conditions. Scale bars = 100 µm. N = 4. *, P<0.05 vs. control.</p