Criterion tgx stain free

The kinase assay was performed with immunoprecipitated HERC1 substrate and purified full-length c-ABL1 (Invitrogen, #P3049) as described elsewhere [44 (link)]. Briefly, the HEK-293T cells were lysed in CHAPS buffer (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.3% CHAPS, 50 mM NaF, 0.5 mM EDTA, 1mM Na₃VO₄, 1mM PMSF, 2 µg/mL leupeptin, 5 µg/mL aprotinin, 2 µg/mL pepstatin), and endogenous HERC1 was immunoprecipitated as mentioned above in Section 4.3. Purified full-length ABL (Invitrogen, #P3049) and immunoprecipitated endogenous HERC1 were resuspended in kinase buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl₂, 1 mM MnCl₂, 10 mM NaF, 1 mM NaVO₃, 5% glycerol, 1% NP-40, 1 mM DTT, 1 mM PMSF) and 10 mM ATP (Sigma). After 30 min at 37 °C, reaction was stopped by the addition of SDS–PAGE buffer, followed by boiling for 10 min at 100 °C. Protein samples were separated on 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, and incubated with anti-HERC1 and anti-phosphotyrosine antibodies. Membranes were then washed and incubated with appropriate peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Dallas, Texas, USA). Specific binding was detected using an enhanced chemiluminescence system (Clarity Western ECL Substrate #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).