Rabbit anti igg

The primary antibodies used in the study included rabbit anti-Profilin1 (1:1000, Abcam, ab50667), rabbit anti-Cdc42 (1:1000, Proteintech, Rosemont, IL, USA), rabbit anti-PAK (1:1000, Proteintech, Rosemont, IL, USA), rabbit anti-Phospho PAK (1:1500, Proteintech, IL, USA), rabbit anti-IgG (1:50, Santa Cruz, Dallas, TX, USA), rabbit anti-JNK (1:1000, Abcam, ab179461, London, UK), rabbit anti-Phospho JNK(1:1000, Abcam, ab124956, London, UK), mouse anti-MyHC (1:100, DSHB, CO, USA), mouse anti-MyoG (1:100, DSHB, Iowa, IA, USA), mouse anti-GAPDH (1:1000, Zhongshan, Golden Bridge Bio-technology, Beijing, China), and mouse anti-tubulin (1:3000, Proteintech, Rosemont, IL, USA). The secondary antibodies included horseradish peroxidase goat anti-mouse/rabbit IgG (1:10000, Zhongshan, Golden Bridge Bio-technology, Beijing, China), PAK inhibitors (Selleck, PF-3758309), and JNK inhibitors (GLPBIO, SP600125). A G-LISA^® Cdc42 Activation Assay Biochem Kit™ (cytoskeleton, Cat. # BK127-S) was used.

Cells were washed with cold PBS and then lysed on ice for 30 min in lysis buffer (50 μL RIPA buffer 2X, 50 μL PBS). The cells were centrifuged at 13000 rpm for 15 min at 4 °C to eliminate cellular debris. 50 μl of cell lysate was collected and 10 μl of GST-Grb2 beads and incubated over night with gentle rocking at 4 °C. Cell extract was mixed with 2X Laemmli and denatured at 95 °C for 5 min. Proteins were separated by migration through a denaturing 8% SDS-PAGE gel and electro-transferred onto a nitrocellulose membrane (Protan). The membrane was blocked by 5% non-fat milk-TBST for 1 h at RT. The proteins were analyzed using a rabbit polyclonal primary antibody anti-TGFβ RII (Santa Cruz biotechnology #sc-220-R) diluted by 1/200 5% BSA-TBST and a rabbit polyclonal primary antibody anti-p85 both incubated overnight at 4 °C. After washing, membranes were incubated with peroxidase-conjugated secondary antibody rabbit anti-IgG (Santa Cruz biotechnology #2005) diluted by 1/5000 in 5% non-fat milk-TBST for 1 h at RT. After washing, membranes were developed using Super Signal detection kit (Thermo).