Herc1 a301 904a

Protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad). 100 µg of total protein were loaded and run onto 4–14% precast gradient gel (Criterion TM TGX Stain-Free TM) electrophoresis and transferred to PVDF (Bio-Rad, Hercules, California, CA, USA) membranes by using the protocol described elsewhere [22 (link)]. Blots were blocked in TBS (Tris-HCl pH7.6, 150 mM NaCl) plus 5% nonfat milk for 1 h at room temperature (RT) and then decorated with appropriate antibodies (HERC1 A301-904A, Bethyl Laboratories, Inc., Montgomery, TX, USA, Tubulin sc-23948, and p44/42 MAPK (ERK1/2) #4696s, phospho-p44/42 MAPK (T202/Y204) #4377s; Vinculin MA5-11690, Sigma-Aldrich; BCR Cell Signaling Tech, phospho-Tyr, sc-7020, Santa Cruz Biotechnology, Dallas, TX, USA; in PBS-Tween 0.5% overnight at 4 °C. Membranes were then washed with PBS-Tween 0.5% 3 times for 15 min each, incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology) for 1 h at RT, and washed again in PBS-Tween 0.5%. Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECLSubstrate #170-5061, Bio-Rad, Hercules, CA, USA).

One hundred micrograms (100 µg) of total protein were loaded and run onto 4%–14% gradient precasted gel (Criterion^TM TGX Stain-Free^TM, Bio-Rad, Hercules, CA, USA) electrophoresis and transferred to PVDF (Bio-Rad, Hercules, CA, USA) membranes as described elsewhere [42 (link)]. Blots were blocked in TBS plus 5% non-fatty acid milk and decorated with appropriate primary antibodies (HERC1 #A301-904A, Bethyl Laboratories Inc., Montgomery, TX, USA; β-Actin, sc-47778, phospho-Tyr, sc-7020, c-Abl, sc-23 and BCR, sc-20707, Santa Cruz Biotechnology, Dallas, Texas, USA; Vinculin MA5-11690, Invitrogen, Carlsbad, CA, USA). Membranes were then washed and incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECL Substrate #170-5061, Bio-Rad, Hercules, CA, USA).