Hrp conjugated goat anti mouse igg h l

Proteins in each group were extracted using radio immunoprecipitation assay lysis buffer (Beyotime Biotechnology, Haimen, China) containing 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology). The concentration of proteins in each group was measured using a BCA Protein Assay Kit (Beyotime Biotechnology). Forth microgramme proteins in each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk, followed by incubating with primary antibodies against FMNL2 (1:500; Novus, Oakville, ON, Canada), E-cadherin (1:2000, Proteintech, Wuhan, China), Vimentin (1:2000, Proteintech), N-cadherin (1:2000, Proteintech) or β-actin (1:500; Bioss, Beijing, China) at 4 °C overnight. After rinsing in Tris buffered saline with Tween (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat-anti-rabbit IgG(H+L) or HRP-conjugated goat-anti-mouse IgG(H+L) (1:5000; Beyotime Biotechnology) at 37 °C for 45 min. After rinsing in TBST, the targeted bands were visualized using an ECL Kit (Beyotime Biotechnology).

MCF-7 cells were harvested, and then lysed in RIPA buffer containing PMSF (Solarbio, R0010) and proteinase inhibitors (Sangon Biotech, C600386-0001), and the lysates were clarified by centrifugation at 12,500 rpm for 15 min at 4°C. A BCA Protein Assay Kit (Beyotime Biotechnology, P0012) was used to determine protein concentration. Total cellular protein was separated by SDS-PAGE and electro-transferred onto PVDF membranes (Millipore, ISEQ00010). Then the membranes were blocked with 5% milk powder (Sangon Biotech, A600669-0250) and washed by 0.1% TBS/T for 10 min. Subsequently, the membranes were incubated with primary antibody. The following primary antibodies were used: mouse monoclonal anti-ESR1 (Santa Cruz Biotechnology, sc-8002), rabbit polyclonal anti-DNAJC12 (Proteintech, 12338-1-AP), rabbit polyclonal anti-ERBB4 (Proteintech, 19943-1-AP), or mouse monoclonal anti-β-actin (Beyotime Biotechnology, AF0003) at 4°C overnight. After washing three times in 0.1% TBS/T, membranes were incubated with the secondary HRP-conjugated goat anti-rabbit IgG (H + L) (Beyotime Biotechnology, A0208) or HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime Biotechnology, A0216). Proteins were visualized with an ECL system.

DMF (purity>99%) was dissolved in 100% dimethyl sulfoxide (DMSO), and the final DMSO concentration in the cell culture did not exceed 0.1% throughout the study. Phycoerythrin (PE)-conjugated anti-mouse CD206 (Clone M1), PE-conjugated anti-mouse CCR7, PE-conjugated anti-mouse MGL1/2, PE-conjugated anti-mouse MHCII, and corresponding isotype controls were purchased from BD Pharmingen (San Diego, CA, USA). Penicillin, streptomycin, DCFH-DA, HRP-conjugated goat anti-mouse IgG (H+L), and MTT were purchased from Beyotime (Haimen, Jiangsu, China). The Annexin-V/PI apoptosis kit was purchased from BD Pharmingen. Matrigel and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Atorvastatin (ATO, catalog number: BD18106) and cyclophosphamide (CTX, catalog number: BD122996) were purchased from Bidepharm (China). Pluronic F127 was purchased from Sigma-Aldrich (United States). ATP Assay Kit (catalog number: S0026) was purchased from Beyotime biotechnology (China). Anti-CD16/32 (catalog number: 65080-1-L), anti-CD3-FITC (catalog number: FITC-65077), and anti-CD8a-APC (catalog number: APC-65069) were purchased from Proteintech (China). Anti-CD274 (PD-L1)-PE (catalog number: 124308) was purchased from Biolegend (United States). Anti-calreticulin primary antibody (catalog number: ab271865) was purchased from Abcam (United States). HMGB1 rabbit monoclonal antibody (catalog number: AG2167), β-actin mouse monoclonal antibody (catalog number: AF0003), Alexa Fluor 488-labeled goat anti-rabbit IgG (H + L) (catalog number: A0423), HRP-conjugated goat anti-mouse IgG (H + L) (catalog number: A0216), and HRP-conjugated goat anti-rabbit IgG (H + L) (catalog number: A0208) were purchased from Beyotime biotechnology (China).