ECM IB4+ t=0 expressed genes reps1-7

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=0 (smapled immediately after selection protocol). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

ECM IB4+ t=24LN expressed genes reps1-6

Data showing genes detected on microarrays for IB4-expressing dorsal root ganglion neurons at t=24LN (sampled after 24 hr culture on laminin-coated substrate). See Read-me file for details. Only genes detected above background are listed; of 144 ECM genes on the arrays, only 36 genes were expressed by both neuron populations

HspB1 is associated with F-actin.

<p>Representative blots showing precipitation of F-actin complexes (with biotinylated phalloidin, A–B) and IPs of HspB1 (C–D) from total cellular lysates (A, C) or the cytoskeletal pellet fraction (B, D). PC12 cells were treated as described in the Methods. Cell cultures were exposed to 10 µM SB203580 for 1 hr, after which cultures were either incubated for an additional 30 mins, or stressed with heat shock at 42°C for 30 mins; control cells were not treated. Immediately after treatments, cells were collected, lysed with actin stabilization buffer; samples were separated for analysis as total cell lysate or further fractionated into the TritonX-100 insoluble cytoskeletal pellet. Samples were incubated with biotinylated-phalloidin followed by precipitation of the captured complexes with streptavidin-linked magnetic beads. Precipitated fractions were then subjected to SDS-PAGE and sequentially immunoblotted to detect pHspB1, total HspB1 and actin. Pulldown of F-actin also captures HspB1 in both the cell lysates (A) and the cytoskeletal fraction (B); similarly the HspB1 IPs also bring down actin and this is enhanced in the cytoskeletal fraction.</p

ECM IB4neg t=0 expressed genes reps1-7

Data for non-IB4 expressing DRG neurons at t=0; these were sampled immediately after the selection protocol that removes the IB4-expressing neurons, leaving the IB4-negative neurons behind. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

ECM IB4neg t=24LN expressed genes reps1-6

Data for non-IB4 expressing DRG neurons at t=24LN; these were sampled after 24 hr culture on laminin-coated substrate. Only genes detected as being expressed are presented.Of 144 ECM genes on the arrays, 36 genes were expressed by both neuron populations

List of genes on microarray ORN-013.2

Complete list of genes on GEArray ORN-013.

List of primary and secondary antibodies, with experimental dilutions, used for immunoblotting (IB), immunocytochemistry (ICC) and immunoprecipitation (IP).

<p>List of primary and secondary antibodies, with experimental dilutions, used for immunoblotting (IB), immunocytochemistry (ICC) and immunoprecipitation (IP).</p

Specificity of the Phalloidin pull-down for F-actin.

<p><i>In vitro</i> preparations (2.5 or 5 µg) of globular actin (G-actin) and filamentous actin (F-actin) were prepared using an <i>in vitro</i> actin–binding assay kit (Cytoskeleton) and incubated with 5 µg of biotinylated-phalloidin as detailed in the Methods section. The resulting solutions were separated into supernatant (non-phalloidin interacting) and pull-down (phalloidin interacting) samples and assayed by Western blotting. Additionally, untreated G- or F-actin samples were also probed. Note that biotinylated-phalloidin selectively precipitates F-actin, but not G-actin, and that this pull-down is reasonably efficient.</p

List of genes on microarray ORN-013

Complete list of genes on GEArray ORN-01

HspB1 is phosphorylated and redistributed to the cytoskeletal fraction during cellular stress.

<p>PC12 cells were treated as described in the Methods, subsequently lysed and separated by cellular fractionation into Triton X-100 soluble (cytosolic; lysate) and Triton X-100 insoluble (cytoskeletal; pellet) samples, or were left as crude total protein samples. Untreated cells (Lanes 1–3); treated with 10 µM SB203580 (Lanes 4–6); heat shocked cells (Lanes 7–9); cells treated with inhibitor and heat shock (Lanes 10–12). Western blots were sequentially probed with antibodies to pS15-HspB1, pS86-HspB1, HspB1 and actin; blots were stripped after each probe and prior to the next. Note the increased amount of HspB1 in the cytoskeletal fraction after HS, as well as the increased amount of phosphorylated HspB1.</p