Stripping solution

Western blots were performed as previously described [5 (link)]. Briefly, whole cell lysates were isolated using RIPA [10 mM Tris base pH 7.2, 150 mM NaCl, 1% Nadeoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)] supplemented with protease inhibitors (Sigma), phosphatase inhibitors (Sigma) and phenylmethanesulfonylfluoride. Lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4 °C. Protein concentrations were de termined using BCA Protein Assay Reagent (Pierce, Rockford, IL) and separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Antibodies were used according to manufacturer’s recommended conditions. Molecular weight markers (Precision Plus Protein Kaleidoscope Standards, Bio-Rad, Hercules, CA) confirmed the expected size of the target proteins. Immunoblots were developed with Luminata Classico or Crescendo ECL (EMD Millipore). Blots were stripped with stripping solution (Bio-Rad) at 37 °C for 15 minutes and then re-probed with selected antibodies. Equal protein loading was confirmed by immunoblotting with antibody to β-actin or GAPDH. Densitometry was performed using Scion Image Program. Each band was normalized to background on the blot, and then normalized to their respective actin band. All bands were compared to the 0 μM treatment group, which was given the value of 1 as previously reported [6 (link)].

Whole-cell lysates were isolated using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich), phosphatase inhibitors (Sigma Aldrich), and phenylmethanesulfonylfluoride (Sigma Aldrich). Lysates were centrifuged at 14 000 rpm for 1 hour at 4°C. Protein concentrations were determined using Pierce^™ BCA Protein Assay reagent (Thermo Fisher Scientific) and separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Antibodies were used according to the manufacturers’ recommended conditions. Molecular weight markers (Precision Plus Protein Kaleidoscope, Bio-Rad) were used to confirm the expected size of the proteins of interest. Immunoblots were developed with Luminata Classico or Crescendo Western HRP Substrate (EMD Millipore). Blots were stripped with stripping solution (Bio-Rad) at 65°C for 20 minutes and then re-probed with selected antibodies. Equal protein loading was confirmed using β-actin. Rabbit monoclonal anti-PIM3 (4165) was from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti-β-actin (A1978) was from Sigma Aldrich (St. Louis, MO).

Radioimmunoprecipitation assay (RIPA) buffer, supplemented with protease inhibitors (Sigma Aldrich, Burlington, MA), phosphatase inhibitors (Sigma Aldrich), and phenylmethanesulfonylfluoride (Sigma Aldrich), was used to isolate whole-cell lysates. Lysates were centrifuged at 14,000 rpm for 30 minutes at 4 °C followed by determination of protein concentrations using Pierce BCA Protein Assay reagent (Thermo Fisher Scientific). Proteins were separated by electrophoresis on Expressplus™PAGE gels (GenScript, Piscataway, NJ). Antibodies were used according to the manufacturers’ recommended conditions. Molecular weight markers (Precision Plus Protein Kaleidoscope, Bio-Rad, Hercules, CA) were used to confirm the expected size of the proteins of interest. Immunoblots were developed with Luminata Classico or Crescendo Western horseradish peroxidase (HRP) Substrate (EMD Millipore). Blots were stripped with stripping solution (Bio-Rad) at 65 °C for 20 minutes and then re-probed with selected antibodies. Equal protein loading was confirmed using β-actin or vinculin as an internal control.

Whole-cell lysates were isolated using radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitor (Sigma), phosphatase inhibitor (Sigma), and phenylmethane-sulfonylfluoride (PMSF) (Sigma) as previously described (15 (link)). Protein concentrations were determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), and samples were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad, Hercules, CA) was utilized to confirm expected size of target proteins. Gel transfer to blotting membrane was completed using Trans-Blot Turbo Transfer System (Bio-Rad) per manufacturer’s protocol. Antibodies were utilized according to the manufacturers’ recommendations. Immunoblots were developed with Immobilon Classico or Crescendo Western HRP Substrate (EMD Millipore, Millipore Sigma, Burlington, MA). Blots were stripped with stripping solution (Bio-Rad) at 65°C for 20 minutes and then re-probed with selected antibodies. Equal protein loading between samples was confirmed using β-actin as an internal control.