Anti c fos

Materials: All chemicals unless otherwise specified were obtained from Sigma-Aldrich.
Antibodies directed against Tubulin (catalog no. T7816), c-Fos (F7799) and Ubc-9 (catalog no. U2634) were obtained from Sigma-Aldrich. Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (catalog no. 39-8600), anti-c-FOS (catalog no. MA5-15055), anti-MMP7 (catalog no. PA5-28076), anti-c-JUN (catalog no. MA5-15172), Horseradish peroxidase (HRPO)conjugated anti-rabbit (catalog no. 65-6120) and HRP-conjugated anti-mouse (catalog no. 62-6520) antibodies were obtained from Invitrogen. Anti-H3 (catalog no. ab1791) and anti-c-FOS (phospho T232) (catalog no. ab193365) were obtained from Abcam. Anti-actin (catalog no. 4970), anti-caspase3 (catalog no. 9662), anti-PIAS1 (catalog no. 3550) and anti-c-JUN (phospho S73) (catalog no. 3270) were obtained from Cell Signaling Technology.

EMSA was carried out according to DIG gel shift kit, 2 nd generation (Roche) kit's protocol. EMSA was done with small fragment of PIAS1 promoter (39 bp) with nuclear extracted proteins of control cells. The probes corresponding to identify AP-1 binding site on PIAS1 promoter, were 3' end labeled with Digoxigenin-11-ddUTP (DIG-dd-UTP) molecule by terminal transferase provided in DIG gel shift kit. The probes were incubated with nuclear extract from control cells. For competition assay 100-fold molar excess of unlabeled probes and nuclear extract were incubated along with labeled probes. For Super shift assay, nuclear extract was pre-incubated with anti c-Fos (Invitrogen, MA5-15055) antibody for 2hrs on end-to-end rotor (360°) at 4°C. After pre-incubation all samples were proceeded for binding reaction at room temperature. All samples were run on 5% native polyacrylamide gel.