Ab179467

RIPA lysis buffer (Biotime, Hangzhou, China) was used to obtain total proteins from BC tissues and cells, and the quality of the protein was determined by BCA kit (Pierce, USA). Proteins were separated by 10% SDS-PAGE, and were subsequentially transferred onto the PVDF membranes (Millipore, MA, USA). The membranes were blocked by 5% non-fat milk, and incubated with the primary antibodies against gp96 (1:1000, #ab227293, Abcam, UK), TSG101 (1:1000, #ab125011, Abcam, UK), p53 (1:2000, #ab1101, Abcam, UK), GAPDH (1:2000, #ab8245, Abcam, UK), CD63 (1:1500, #ab271286, Abcam, UK) and β-actin (1:2000, #ab179467, Abcam, UK) at 4°C overnight. In the second day, the membranes were probed with the secondary antibodies for 1h at room temperature, and an ECL system (Bio-Rad, CA, USA) was employed to visualize the protein bands. The expression levels of the proteins were normalized by GAPDH.

RIPA buffer and protease and phosphatase inhibitors were used to extract proteins from cells according to their instructions. The protein levels of target molecules were tested by SDS-PAGE, and finally evaluated with a Bio-Rad system. The primary antibodies used for Western Blots were anti-β-actin (1:2000), abcam, ab179467 and anti-MCL-1, abcam, ab32087 (1:1000).

We utilized cell lysis buffer (Beyotime), containing 1% PMSF (Amresco), to cleave proteins for 30 minutes on ice. Then, we centrifuged the lysed cells at 12 000 g for 10 minutes at 4°C to extract the supernatant for protein quantification. We utilized the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) to quantify protein concentration. The obtained supernatant was then boiled for 10 minutes by adding 5X SDS. Protein (50 μg) was added to the prepared 12% SDS‐PAGE gels for electrophoretic separation and transferred to 0.45 µm PVDF membranes (Amersham Hybond, GE Healthcare). We then utilized 1% albumin from bovine serum (Amresco) to block the PVDF membranes for 2 hours. Then, the membranes were incubated overnight with diluted xCT (1:1000, ab37185; Abcam) and β‐actin (1:1000, ab179467; Abcam) antibodies on a shaker at 4°C. We washed the membranes with TBS‐T (0.1% Tween‐20) at room temperature three times for 10 minutes. The goat anti‐rabbit IgG H&L (HRP) (1:2000, ab7090; Abcam) for 1 hour was utilized to incubate the membranes. After washed, the membranes were exposed to enhanced chemiluminescence substrate detection solution (Lulong Biotech) subsequently.

The abundance of perforin in the tissues was detected by western blot. In short, the total proteins in the tissues were extracted by RIPA lysis buffer (Beijing Nobleide Technology Co., Ltd., Beijing, China). The total proteins were quantified and then separated with SDS-polyacrylamide gel. After that, the proteins were transferred to polyvinylidene fluoride membranes by wet-transfer methods. Subsequently, the membranes were blocked with 5% fat-free milk for 2 hours and then were incubated with the primary antibodies including anti-perforin (ab256453, Abcam), Occludin (ab216327, Abcam), Claudin-5 (ab131259, Abcam), and anti-β-actin (ab179467, Abcam) at 4°C overnight. The membranes were washed with the TBST for three times and then were incubated with secondary antibodies for 2 hours. Finally, the proteins were quantified by a chemiluminescence detection system.

Cells were washed, lysed, and processed as previously described (Liu et al., 2020 (link)). Equal amount of proteins for each sample were separated by 10% SDS-polyacrylamide gel and then transferred to Nitrocellulose (NC) membrane for 70 min at 100 V. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 (0.1%) (TBS-T) for 1hr at room temperature. Rabbit anti-TNFR2 antibody (Abcam, MA, United States, ab109322) were used at 1:1000 dilution at 4°C overnight. β-Actin was detected using anti-rabbit antibody (Abcam, MA, United States, ab179467) as reference protein. Membrane was rinsed with TBS-T for 10 min, five times and incubated with 1:5000 goat anti-rabbit-Alexa Fluor 680 or donkey anti-mouse-Alexa Fluor 680 (Molecular Probes, MA, United States) for 1 h at room temperature. The blots were scanned using an Odyssey fluorescence scanner (LI-COR Biosciences, NE, United States).

The cells were lysed for 20 min in 180 μL of lysis buffer (Beyotime, Shanghai, China) on ice before the centrifugation at 4 °C and 14,000 rpm for 10 min. The total protein concentration was estimated using a bicinchoninic acid (BCA) protein detection kit (CWBIO, Shanghai, China) with bovine serum albumin (BSA) as the standard. The proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After being blocked with 5% (w/v) skimmed milk for 2 h, the membranes were probed with corresponding primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h. The primary antibodies used were: TLR4 (1:1000, Abcam, ab13556), myeloid differentiation factor 88 (MyD88, 1:1000, Abcam, ab133739), nuclear factor kappa-B (NF-κB) p65 (1:1000, Abcam, ab32536), NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), claudin-1 (1:2000, Abcam, ab211737), occludin (1:1000, Abcam, ab216327), ZO-1 (1:1000, Abcam, ab276131), myosin light-chain kinase (MLCK, 1:1000, ABclonal, A3835), and β-actin (1:5000, Abcam, ab179467). The protein intensity was quantified using the Image J software (version 1.8.0, NIH, Bethesda, MD, USA).