Skim milk

The cells at a concentration of 5 × 10⁵cells/ml were seeded in 100 mm Petri dishes and incubated to attain confluence. The cellswere exposed to the inhibitory concentration of GAE (1.5 mg/ml) for 24 h. The cells without GAE treatment represented the control group. The medium was removed and the cells were washed twice with ice-cold PBS and lysed with RIPA buffer. The cell lysate was centrifuged (14,000 rpm, 10 min) and the supernatant was stored at − 20 °C till further use. The protein content of the cell lysate was quantified by Lowry’s method. 50 μg of protein was separated in 10% SDS- PAGE and the proteins were transferred onto a nitrocellulose membrane (BioRad, USA) and blocked with 5% skim milk (BioRad, USA). Following this, the membrane was exposed to primary antibodies (4 °C, 24 h) against Bax (1:500 Abcam, USA) ,Bcl-2(1:500 Abcam, USA), Bcl-XL (1:500 Abcam, USA), Caspase 3 (1:200 Abcam, USA), PI3K (1:500 Abcam, USA), p PI3k(1:1000 Abcam, USA), Akt(1:500 GenetexBio, USA), p Akt(1: 1000 GenetexBio, USA) and β actin (1:1000 1:1000 Abcam, USA). The membrane was then incubated with HRP-conjugated anti-mouse (1:2500) and anti-rabbit (1:2000) secondary antibodies for 1 h (RT) with continuous shaking. The protein bands were visualized using an ECL staining kit (Amersham Pharmacia Biotech, Sweden).

Breast tissue extracts were electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrotransferred to polyvinylidene fluoride (Solarbio) membranes. The membranes were sealed with 1X phosphate-buffered saline, 0.1% Tween-20, and 5% skim milk (Bio-Rad) and then incubated with primary antibodies at 4°C overnight. Membranes were then washed with 1X TBS-0.1% Tween-20 film and incubated with secondary antibodies at room temperature for 1 h. Autoradiography was performed with a Bio-Rad ChemiDoc XRS+ system.

After washing with PBS, cells were lysed with the pre-chilled lysis buffer. Later, the collected cell lysates were subjected to 15 min of centrifugation at 14,000 × g and 4°C and boiling with 5 × sample buffer (BSA; Thermo Fisher Scientific, Inc., CA, United States) after the protein content was measured. Afterward, WB assay was performed on those protein samples. To carry out WB, the 4%–20% precasting gel (Bio-Rad Laboratories) was used to transfer protein onto the nitrocellulose membranes, and then 5% skim milk was utilized to block the membranes (Bio-Rad Laboratories) for 1 h under ambient temperature. Later, specific antibodies (dilution, 1:1,000), including anti-SLC17A9, anti-VEGF, anti-Bax, anti-Ki67, anti-MTA1, anti-Bcl-2, anti-MMP-2, and GAPDH (Santa Cruz, CA, United States), were used to block membranes. Subsequently, secondary antibodies (Santa Cruz) were utilized to incubate membranes at ambient temperature for 1 h, followed by visualization using the ECL solution (Bio-Rad Laboratories). Finally, images were taken using the chemiluminescence imaging system (Mini HD9; UVitec, Cambridge, United Kingdom).