Rabbit anti human rack1 antibody

Western blot was performed according to the standard procedures. Briefly, tissues and cell lysates were extracted using RIPA buffer (Promega, USA), the protein concentrations were determined by a BCA™ Protein Assay Kit (Pierce, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto PVDF membranes (Merk-Millipore, USA). The membranes were first incubated with indicated primary antibodies overnight (4 °C), and then followed by HRP-conjugated secondary antibodies for 2 h at room temperature. Blots were detected with ECL Western Blotting Substrate (Promega, USA) and quantized by Image J software (NIH, USA), β-actin was used as an internal control. The following antibodies were included: a rabbit anti-human RACK1 antibody (Abcam, USA, 1:500 dilution), a mouse anti-human β-actin antibody (Abcam, USA, 1:1,000 dilution), a horseradish peroxidase (HRP)-conjugated IgG (Abcam, USA, 1:2,000 dilution).

Briefly, 2×10⁵ cells were plated into 24-well plates with a round slide in each well and cultured at 37 °C with 5% CO₂ overnight. After treated for 48 h as described above, the cells were fixed with 4% paraformaldehyde for 30 min at 4 °C, rinsed with PBS, and incubated with 0.5% Triton X-100 for 10 min at room temperature, incubated with the block solution for 30 min at 4 °C, incubated with a rabbit anti-human RACK1 antibody (1:50 dilution) (Abcam, USA) at 4 °C overnight, rinsed with PBS, incubated with IgG-TRITC (1:1,000 dilution) (Abcam, USA) for 2 h at room temperature, rinsed with PBS again and followed by Hoechst 33258 staining (Sigma-Aldrich, USA) for 10 min. Finally, the cells were mounted and observed under an immunofluorescence microscopy protect from light.