Ab223500

To measure the protein level of MAT2B in human breast cell lines and explore whether AKT and ERK signalling pathways were associated with MAT2B knockdown in TNBC, we performed western blot analysis. We divided MDA-MB-231 cells into four groups as described above. Proteins were extracted, loaded, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked, washed, and incubated overnight with primary antibodies against MAT2B (1:500 dilution;ab109484;Abcam), phosphorylated AKT (p-AKT; 1:1,000 dilution; ab38449; Abcam), AKT (1:1,000 dilution; ab8805; Abcam), phosphorylated ERK1/2 (p-ERK1/2; 1:1,000 dilution; ab223500; Abcam), ERK1/2 (1:1,000 dilution; ab17942; Abcam), and actin (1:2,000 dilution; sc70319;Santa Cruz). The membrane was then washed, incubated with secondary antibodies (Santa Cruz Biotechnology), and visualised by enhanced chemiluminescence. Band intensity was quantified with ImageJ software and protein expression was standardised to actin.

Tissues or cells were lysed in RIPA lysis buffer (Beyotime) to extract total proteins. After separation by 12% SDS‐PAGE, proteins were transferred onto PVDF membranes (Bio‐Rad) and then blocked in 5% skim milk. The membranes were next incubated with the primary and secondary antibodies, including anti‐MAP4K3 (#92427; Cell Signaling Technology), antiproliferating cell nuclear antigen (anti‐PCNA; ab18197; Abcam), anti‐Bcl‐2‐associated X protein (anti‐Bax; ab182733), anti‐ERK1/2 (ab17942; Abcam), anti‐p‐ERK1/2 (ab223500; Abcam), anti‐JNK (AB208035; Abcam), anti‐p‐JNK (ab76572; Abcam), anti‐p38 (ab32142; Abcam), anti‐p‐p38 (ab178867; Abcam), anti‐GAPDH (ab9485; Abcam) and goat‐anti rabbit (ab205718; Abcam). The protein signals were detected using an enhanced chemiluminescence system (Beyotime).

The CAL-27 cells were collected and treated with radio-immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor. Cell samples were quantified by a bicinchoninic acid (BCA) kit (Beyotime). Then, the total proteins were separated by a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After sealing with 5% non-fat milk in Tris buffered saline Tween (TBST), the membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies used were as follows: Ki67 (ab16667, Abcam, UK), PCNA (#2586, cell signaling technology, CST, USA), caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), survivin (ab76424, Abcam), SOX2 (ab97959, Abcam), NANOG (ab109250, Abcam), OCT4 (ab18976, Abcam), STAT3 (ab119352, Abcam), p-STAT3 (ab76315, Abcam), ERK1/2 (ab17942, Abcam), p-ERK1/2 (ab223500, Abcam). On the next day, the horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (#7076, CST) or anti-rabbit IgG (#7074, CST) were incubated for 2 h at room temperature. Finally, the targeted strips were visualized using an electrochemiluminescence (ECL) solution. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

After transfection for 48 h, radioimmunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors was used for total protein extraction. Proteins from each group were separated and transferred to a polyvinylidene fluoride membrane. The membrane was then blocked, followed by incubation with primary antibodies (1:1000) overnight at 4 °C. Next, the secondary antibodies (1:5000) were given for 1 h at room temperature. After incubation with the enhanced chemiluminescence (ECL) reagent, the expression values of proteins were normalized against GAPDH and quantified with QUANTITY ONE software. Anti-human SIL1 (1: 500, ab5639), Cyclin D1 (1: 2000, ab16663), CDK4 (1: 2000, ab108357), CDK6 (1: 1000, ab124821), ERK1/2 (1: 1000, ab17942), p-ERK1/2 (1: 500, ab223500), Acitve-Caspase9 (1: 1000, ab32539), Bcl-2 (1: 1000, ab182858), Bax (1: 1000, ab32503), and GAPDH (1: 5000, ab181602) were all purchased from Abcam (USA). Each experiment included triplicate measurements. Secondary antibodies, Goat Anti-Mouse IgG H&L (HRP) (ab205719), and Goat Anti-Rabbit IgG H&L (HRP) (ab6721) were also purchased from Abcam (USA).