Gapdh 60004 1 lg

Antibodies for GSDME (ab215191), GSDMD (ab209845) were purchased from Abcam (Cambridge, Cambridgeshire, Britain). Antibodies for caspase-1 (3866T), caspase-9 (9502T), caspase-3 (9668T), caspase-8 (9746T) and γH2AX (9718T) were purchased from Cell Signaling Technology (Danver, MA, USA), and GAPDH (60004-1-lg) was from Proteintech Group (Wuhan, Hubei, China). The secondary antibodies including HRP Goat Anti-Mouse IgG (H+L) (AS003), HRP Goat Anti-Rabbit IgG (H+L) (AS014) and FITC Goat Anti-Rabbit IgG (H+L) (AS011) were bought from Abclonal (Wuhan, Hubei, China).
Other reagents were purchased as follows: caspase-3 inhibitor (Z-DEVD-FMK) (HY-12466) and ROS scavenger N-acetyl cysteine (NAC) (HY-B0215) were from MedChemExpress (Monmouth Junction, NJ, USA); protease inhibitor cocktail tablets (BL612A) were from Biosharp (Beijing, China).

Antibodies against the following proteins were used: SMYD2 (ab195365), STAT3 (ab68153), phospho-STAT3 (Y705, ab76315), DKK3 (ab186409), and P-gP (ab170904) from Abcam; β-tubulin (10068-1-AP), CDK6 (14052-1-AP), N-cadherin (22018-1-AP), and GAPDH (60004-1-lg) from Proteintech; and E-cadherin (11A24) and vimentin (BM0135) from Boster (Wuhan, China). The following reagents were used in this research: AZ505 (HY-15226), cisplatin (HY-17394), and sunitinib (HY-10255A) from MedChemExpress (Monmouth Junction, NJ, USA); and doxorubicin (A3966), 5-fluorouracil (5-FU) (A4071), and docetaxel (A4394) from APExBIO (Boston, MA, USA).

Antibodies against P-p38 MAPK (4511), p38 MAPK (9212), and P-MKK3/6 (12280) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against the HCV core protein (C7-50) (sc-57800) and TAB1 (B-3) (sc-166138) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Antibodies against HA (66006-2-lg and 51064-2-AP), Flag (20543-1-AP), TAB1 (27566-1-AP) and GAPDH (60004-1-lg) were obtained from ProteinTech Group (Wuhan, China). Alexa Fluor 488 (34106ES60), and Cy3 (33108ES60) were purchased from YEASEN (Shanghai, China). SB203580 (S1076) was purchased from Selleck Chemicals (Houston, TX). Dynabeads® Protein G (10004D) was from Invitrogen (Waltham, Massachusetts).

MDA-MB-231 human TNBC cell line was obtained from the Key Laboratory of Breast Diseases in Jiangxi Province and cultured in high glucose DMEM with 10% fetal bovine serum (FBS). DMEM was purchased from Solarbio (Beijing, China), while FBS was purchased from Gibco (New York, USA). TRX (purity>98%, CAS:127-22-0) was purchased from Herbest Biochemical Technology Co., Ltd. (Baoji, China) and then dissolved in dimethyl sulfoxide (DMSO) from Macklin (Shanghai, China) to prepare a 20 mmol/L (mM) stock solution for storage at -40°C. tBHQ (purity>99%, CAS:1948-33-0) was purchased from MedChemExpress (Shanghai, China) and dissolved in DMSO to prepare a 10 mM stock solution for storage at -40°C.
Primary antibodies against epithelial-cadherin (E-cadherin, ab40772), neural-cadherin (N-cadherin, ab76011), Vimentin (ab92547), and Slug (ab85936) were purchased from Abcam (Cambridge, UK), while phospho-ERK1/2 (28733-1-AP), ERK1/2 (11257-1-AP), and GAPDH (60004-1-lg) were purchased from Proteintech (Chicago, USA).

The proteins of tissues and cells were lysed in M-PER Protein Extraction Reagent (Pierce) mixed with protease inhibitor cocktail. BCA assay kit (Pierce) was used to determine the protein concentrations. The proteins were then separated on 8% SDS-PAGE, and transferred onto nitrocellulose membranes. After blocked with 5% non-fat milk for 1.5 h at room temperature, the membrane were incubated with primary antibodies overnight at 4°C, and followed by incubation with the corresponding secondary antibodies for 1.5 h. The blots were detected by ECL chemiluminescence kit (Millipore). These primary antibodies were obtained from Cell Signaling Technology (Danvers, USA), FOXK1 (# 12025), mTOR (# 2983), p-mTOR (# 5536), AKT (# 4691), p-AKT (# 4060), Cyclin D1(# 2922), Cyclin E1 (# 4129), E-cadherin (# 3195), N- cadherin (# 13116), CDK4 (# 12790), CDK6 (# 3136), and anti-Rabbit IgG-HRP (# 7074). The antibody against vimentin (#ab8979) was purchased from Abcam (Cambridge, USA). The antibody against Gapdh (# 60004-1-lg) and anti-Mouse IgG-HRP (# SA00001-1) were purchased from Proteintech (Rosemont, USA). Selected blots were quantified by using Image J (NIH, USA).

The preparation protocol of CP referred to our previous method (15 (link)). Antibodies against zonula occludens-1 (ZO-1, A11417), Claudin-1 (A11530), Occludin (A2601) and β-actin (AC026) were purchased from ABclonal (Wuhan, China). Antibodies against mucin-2 (MUC2, GB111965) was purchased from Servicebio (Wuhan, China). DSS for colitis model was purchased from MP Biomedicals (molecular weight: 36–50 kDa, Santa Ana, CA, United States). iNOS (18985-1-AP) and CD206 (60143-1-lg) antibodies for immumohistochemical staining were from Proteintech (Wuhan, China). CD80 (16-10A1) antibody for FACS analysis was from Biolegend (San Diego, CA, United States). Polystyrene latex beads with 2 μm diameter were from Sigma Aldrich (Shanghai, China). Antibodies against P65 (8242T) and p-P65 (3033T) used for western blot were purchased from Cell Signaling Technology while GAPDH (60004-1-lg) was from Proteintech (Wuhan, China). Lipopolysaccharide (LPS, L4391) was from Sigma Aldrich (Shanghai, China).

Total proteins were extracted from cells or tissues using the radioimmunoprecipitation assay (RIPA) lysis buffer (P0013C, Beyotime, China) containing protease inhibitor cocktail (04693132001, Roche, Mannheim, Germany). Equal protein concentrations were separated on NuPAGE gels (Invitrogen, Carlsbad, CA, USA) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in tris-buffered saline solution with 0.1% Tween-20 (TBS-T) solution at room temperature for 2 h and then incubated overnight with anti-SNX17 (10275-1-AP, Proteintech, Rosemont, IL, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-lg, Proteintech, Rosemont, IL, USA) antibodies at 4°C. The membranes were subsequently incubated with corresponding secondary antibodies, namely, donkey anti-rabbit immunoglobulin G (IgG) (H + L) (ab186693, Abcam, Cambridge, UK) and donkey anti-mouse IgG (H + L) (ab186699, Abcam, Cambridge, UK) for about 2 h at room temperature and then subjected to detection on the Odyssey IR Imaging System (Li-Cor, Lincoln, Nebraska, USA). Intensity of immunoblots was analyzed using Quantity One (Bio-Rad, Hercules, CA, USA), while relative protein levels were quantified using (ImageJ software).