Mass spectrometry identification of actin as a binding partner of RACK1.

<p><b><i>A</i></b> SH-SY5Y cells were treated with 10 μM FSK for 30 min, RACK1 was IPed from cell lysate and proteins were resolved by SDS-PAGE which was stained with Deep Purple™ to visualize proteins. Gel slices from both RACK1 and control IgG IPs were digested in-gel and the resulting peptides were submitted to tandem mass spectrometry (MS/MS) sequencing for protein identification, n = 2. <b><i>B</i></b> Identification of actin as a putative RACK1 binding protein. β-actin (ACTB), γ-actin (ACTG), alpha skeletal muscle actin (ACTS), aortic smooth muscle actin (ACTA), alpha cardiac muscle actin (ACTC) were identified in the RACK1 IP. A sum of score of peptides that were matched to the best hit within the family of homologous protein sequences (^<b>1</b>). Calculation of the percent sequence coverage included all peptides matched to the protein, i.e., both unique and common peptides (^<b>2</b>). Differentiation between ACTC, ACTS and ACTA proteins was not possible on the basis of the data since the identified peptides were common to all of them (^<b>3</b>). Differentiation between β-actin and γ-actin proteins was not possible on the basis of the data since the identified peptides were common to both isoforms (^<b>4</b>).</p

Activation of the cAMP pathway increases the association of β-actin with <i>BDNF</i> promoter IV via RACK1.

<p>SH-SY5Y cells were treated with vehicle or with 10 μm FSK for the indicated time points, then lysed for a ChIP assay with normal IgG or anti- β-actin antibody. The β-actin-associated <i>BDNF</i> promoter IV (PIV) in the ChIP samples was detected by semi quantitative PCR (<b><i>A</i></b>) and quantitative PCR (<b><i>B</i></b>). <b><i>A</i></b> The level of PIV in the input was analyzed in parallel. The histogram depicts the mean ratio of β-actin -associated PIV to input PIV expressed as percent control ± SEM. One-way ANOVA detected a significant effect of the treatment [P = 0.020]. *p<0.05 (0 min vs. 15 min) and #p<0.05 (15 min vs. 60 min), Newman-Keuls post-hoc analysis. <i>B</i> The table depicts the quantity of <i>BDNF</i> PIV in the β-actin, IgG control or Protein G ChIP samples, expressed as percent of input ± SEM. One-way ANOVA detected a significant effect of the treatment [P = 0.041]. *p<0.05 (0 min vs. 15 min), Newman-Keuls post-hoc analysis. <i>C</i> SH-SY5Y cells were infected with control adenovirus (shCT) or adenovirus expressing shRACK1 for 3 days before treatment with 10 μM FSK for the indicated time points. A ChIP assay was then conducted as described in <b><i>A</i></b>. A n = 6, B n = 4 and C n = 2 per group.</p

Disruption of actin filaments using cytochalasin D attenuates cAMP-mediated <i>BDNF</i> transcription without blocking the phosphorylation of CREB.

<p><b><i>A</i></b> SH-SY5Y cells were incubated with vehicle or 1 μM cytochalasin D for 15 min prior to treatment with 10 μM FSK for 1 h. <i>BDNF</i> and <i>GAPDH</i> mRNA levels were analyzed by RT-PCR. Histogram depicts the mean ratio of <i>BDNF</i> to <i>GAPDH</i> expressed as percent control (vehicle) ± SEM. Two-way ANOVA shows an interaction between FSK and cytochalasin D [P = 0.049]. ***p<0.001, *p<0.05, ns p = 0.177, Newman-Keuls post-hoc analysis. <b><i>B</i></b> SH-SY5Y cells were incubated with vehicle or 1 μM cytochalasin D for 15 min prior to treatment with 10 μM FSK for 30 min, then lysed in IP buffer. Proteins were resolved by SDS-PAGE and the phosphorylation of CREB on serine 133 was examined by western blot analysis. Histogram depicts the mean ratio of phospho-CREB to total CREB, expressed as percent control (vehicle) ± SEM. Two-way ANOVA shows an effect of FSK [P < 0.001] but no effect of cytochalasin D [P = 0.756] and no interaction [P = 0.966]. Subsequent analysis by the method of contrasts (two-tailed unpaired t-test) detected a significant difference between control and FSK in both vehicle and cytochalasin D groups. **p = 0.002 and ***p<0.001. A n = 9, B n = 3.</p

Activation of the cAMP pathway increases the association between RACK1 and β-actin.

<p><b><i>A</i></b> SH-SY5Y cells were treated with vehicle or 10 μM FSK for the indicated duration, then lysed in IP buffer. RACK1 was IPed from whole cell lysate and proteins were resolved by SDS-PAGE. The presence of RACK1 and β-actin was determined by western blot analysis. <b><i>B</i></b> Quantification of <b><i>A</i></b>. Histogram depicts the mean ratio of β-actin to RACK1, expressed as percent control (0 min) ± SEM. One-way ANOVA showed a significant effect of treatment [P = 0.002]. *p<0.05, **p<0.01 vs. 0 min, Newman-Keuls post-hoc analysis. n = 3.</p

β-actin is a direct binding partner of RACK1.

<p><b><i>A</i></b> SH-SY5Y cells were treated with 10 μM FSK for 30 min, and were then lysed in IP buffer. RACK1 was IPed from whole cell lysate and the co-IPed proteins were resolved by SDS-PAGE. Endogenous RACK1 and β-actin were analyzed by western blot analysis. <b><i>B</i></b> Recombinant MBP and MBP-RACK1 were immobilized on an amylose resin column and incubated with SH-SY5Y lysate previously treated with 10 μM FSK for 30 min. After washing, bound proteins were eluted 3 times with 50 mM maltose (E1, E2 and E3) or with loading buffer (MBP and MBP-RACK1 beads). Proteins were resolved by SDS-PAGE and the presence of β-actin and GAPDH was determined by western blot (right panel). The amount of MBP and MBP-RACK1 recovered after elution was also controlled with colloidal coomassie blue staining (left panel). n = 2. <b><i>C</i></b> The experiment was conducted as in panel <b><i>B</i></b> except that immobilized recombinant MBP and MBP-RACK1 were incubated with pure non-muscle actin. After washing, proteins were eluted twice with 50 mM maltose (E1 and E2) or with loading buffer (bead lanes). Eluted proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane and stained with Ponceau S to control the amount of MBP and MBP-RACK1 (upper panel). The presence of actin was subsequently determined by western blot analysis using a pan actin antibody (lower panel). <b><i>D</i></b> SH-SY5Y cells were incubated with vehicle or 1 μM cytochalasin D (cytoD) for 30 min, and then lysed in IP buffer. RACK1 was immunoprecipitated from whole cell lysate and proteins were resolved by SDS-PAGE. RACK1 and β-actin were revealed by western blot analysis. Histogram depicts the mean ratio of β-actin to RACK1, expressed as percent control ± SEM. Two-tailed unpaired t-test, p = 0.40. A n = 3, B and C n = 2, D n = 6.</p

Nuclear association of RACK1 with β-actin.

<p>SH-SY5Y cells were treated with 10 μM FSK for 30 min, nuclear proteins were isolated and nuclear RACK1 was immunoprecipitated. Proteins were resolved by SDS-PAGE and the gel was stained with Deep Purple™ to visualize proteins. The gel slice indicated by an arrow was in-gel protein digested and resulting peptides were submitted to mass spectrometry (MS/MS) sequencing for protein identification. <i>B</i> Table shows the data leading to the identification of β-actin (ACTB) and γ-actin (ACTG) proteins. Numbers refer to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160948#pone.0160948.g001" target="_blank">Fig 1B</a>.</p