Chemidoc xrs electrophoretic imaging system

Total proteins were extracted from cells or tissues using the M-PER mammalian protein extraction reagent (Thermo Pierce, Rockford, USA) and measured using the BCA Protein Assay (Thermo Pierce). Protein samples (50 μg per assay) were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4 °C in the presence of primary antibodies. The primary antibodies for p21, p53, KHSRP, c-Myc, EPCAM, and β-actin were purchased from Cell Signaling Technologies (CST, Danvers, MA, USA), p-c-Myc antibody was from Santa Cruz (CA, USA), and IκBα, Rb1, STAT3, histone-H3, SUMO1, and SUMO2/3 antibodies were from the Proteintech Company (Chicago, IL, USA). The membranes were washed 3 times in Tris-buffered saline with Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibody (CST, MA, USA) for 1 h at room temperature (RT). After washing 3 times with 15 mL of TBST, Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, USA) was added, and the bands were imaged with the Chemidoc XRS⁺ electrophoretic imaging system (Bio-Rad, Hercules, CA, USA). Density scanning of each protein band was performed using Image Lab software (Bio-Rad, Hercules, CA, USA).

Western blotting was performed as previously described 28 (link). The primary antibodies for PD-L1 (CST #13684), IRF1 (CST #8478), STAT1 (CST #14994), p-STAT1 (CST #9167), GZMB (CST #17215), β-actin (CST #4970) and the matching horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Cell Signaling Technologies (CST). Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica) was added, and the bands were imaged with the Chemidoc XRS+ electrophoretic imaging system (Bio-Rad). Density scanning of each protein band was performed using Image Lab software (Bio-Rad). The results were normalized to β-actin.

Cells were collected at 24, 48 and 72 hrs after treatment, rinsed twice with ice-cold PBS, harvested, lysed in Pierce RIPA buffer (Thermo Scientific, MA, USA). Cell lysates were clarified by centrifugation at 12000 × g for 30 min at 4°C and protein concentrations were determined using the BCA protein assay reagent (Pierce, MA, USA). Cell lysates (protein concentration was adjusted by adding certain amount of RIPA buffer) were subjected to 12% sodium dodecyl sulfate-polyacrylamide electophoresis (SDS-PAGE) gels, separated by SDS-PAGE and electrophoretically transferred to Invitrolon polyvinylidene difluoride (PVDF) membranes (Life technologies, CA, USA). Membranes were blocked with 5% skim milk and then incubated overnight with the appropriate primary antibodies (Seajet Scientific Inc, Beijing, China) followed by matching horseradish peroxidase-conjugated secondary antibodies. After washing each sample three times for 10 min with 15 ml TBST, immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA) was added and the result was tested with ChemiDoc XRS⁺ electrophoretic imaging system (Bio-Rad Laboratories, Berkeley, USA).