Beta actin

The total protein from frozen tissues and breast cancer cell lines was purified using RIPA Buffer. Per sample, 30mg of protein was loaded into a denaturing polyacrylamide gel containing SDS and transferred to a metha- nol-activated PVDF filter membrane (Bio-Rad, Hercules, CA, USA). Before immunodetection, membranes were blocked with 5% non-fat dry milk. Primary antibodies, anti-HOXA11 (1:300; Cat#ab72591, Abcam, Cambridge, MA, USA) were diluted in the blocking buffer and incubated at 4 overnight. After subsequently washing with TBST, the membranes were incubated with secondary antibody for 2 hours at room temperature. Beta-actin (Cat#TA-09, ZSGB-Bio) was used as internal reference protein. The experiment was repeated in triplicate. The bands were visulized by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China).

Cells were lysed in RIPA buffer (Beyotime, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The protein concentration was quantified using a BCA protein assay kit (Thermo Scientific, USA). Equal amounts of protein were denatured with loading buffer (Beyotime, China) at 100°C for 10 min, subjected to 12% SDS-PAGE, and then blotted to a methanol-activated PVDF membrane (Millipore, USA). After blocked with 5% bovine serum albumin (ABCONE, China) in tris-buffered saline (TBS) for 1 h at room temperature, the membranes were respectively incubated overnight with the following antibodies at 4°C: MAVS (1 : 1000, Abcam, USA); pho-IRF3 (1 : 1000, Abcam, USA); caspase-3 (1 : 1000, Cell Signaling Technology, USA); IFN-I, IRF3, cyto C, Bax, and Bcl2 (1 : 1000, Affinity Biosciences, USA); and beta-actin (1 : 1000, ZSGB-BIO, China). After being washed for three times with TBS, the membranes were incubated with the horseradish peroxidase- (HRP-) labeled secondary antibody (1 : 2500, ZSGB-BIO, China) for 1 h at room temperature.

Total protein was extracted using lysis buffer (Beyotime Biosciences) containing PMSF (Beyotime Biosciences) and phosphatase inhibitor cocktail (Biotool, Shanghai, China) and then quantified according to the Bradford method. Total protein samples (60 μg) were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore) and incubated in TBST with 5% skim milk (BD Biosciences) at 37°C for 2 h and then different primary antibodies overnight at 4°C. The membranes were washed three times with TBST for 10 min each time and incubated with peroxidase‐conjugated antimouse or anti‐rabbit IgG (1:2000; ZSGB‐BIO) at 37°C for 2 h. Protein bands were visualized with a bio‐imaging system (DNR), and relative protein expression was determined using GAPDH or beta‐actin as a loading control. Thrombopoietin (ab196026) antibodies were purchased from Abcam. Cyclin E1 (#4129), cyclin E2 (#4132), RhoA (#2117), RhoC (#3430), MYC (#2278), EGFR (#4267), P‐EGFR (Tyr1068; #3777), P‐mTOR (Ser2448; #5536), mTOR (#2983), P‐AKT (Ser473; #4060) and AKT (#4865) antibodies were purchased from Cell Signaling Technology. CDK2 (10122‐1‐AP), P27 (25614‐1‐AP), C‐Myc (10828‐1‐AP), beta‐actin (60008‐1‐ Ig) and GAPDH (60004‐1‐Ig) antibodies were purchased from Proteintech. All experiments were repeated independently at least three times.