Goat anti mouse igg

Cells affixed to slides were incubated in a fresh medium containing 200 μM TMZ/DMSO for 48 h. After three washes with PBS, cells were fixed with 4% paraformaldehyde and then left in 0.2% Triton for 10 min to disrupt the cell membrane. 200 μL of blocking solution was added to each well and the cells were treated for 1 h at room temperature. Anti-GINS2 antibody (Proteintech, #16247-1-AP, 1:200) and anti-γH2AX antibody (Abmart, #M63324S, 1:200) was used to culture the cells overnight at 4 °C. Goat Anti-Rabbit IgG (Dylight 594, Abbkine, A23420, 1:200) and Goat Anti-Mouse IgG (DyLight 488, Abbkine, A23210, 1:200) were used to conjugate the corresponding primary antibodies for 1 h. After washing again, the nuclei were stained with DAPI for 30 min and sealed with neutral gum. Fluorescence and colocalization were observed with laser scanning confocal microscopy (Leica, TCS SP8 SR).

Equal amounts of proteins (~40 μg) were separated on 9-11% SDS-PAGE gels and transferred to PVDF membranes. After blocking in 5% skimmed milk, membranes were probed overnight at 4°C with primary antibodies including anti-AMPK (#2532, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-AMPK (#2535, Cell Signaling Technology), anti-NLRP3 (#15101, Cell Signaling Technology), anti-caspase-1 (#22915-1-AP, Proteintech, Chicago, IL, USA), anti-IL-1β (#sc-12742, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (#A01010, Abbkine, Redlands, CA, USA). Membranes were washed 3 times in Tris-buffered saline with 0.1% Tween 20 and subsequently labeled with the HRP-conjugated goat anti-rabbit IgG (#A21020, Abbkine), goat anti-mouse IgG (#A21010, Abbkine), or mouse anti-Armenian hamster IgG (#sc-2789, Santa Cruz Biotechnology) at a dilution of 1 : 10000. Blots were imaged on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China) and analyzed using ImageJ2x software.

Ginsenoside Rh4 (Figure 1A, purity ≥ 99%) was purchased from Puruifa Technology Development Co., Ltd. (Chengdu, China). Roswell Park Memorial Institute-1640 (RPMI-1640) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific). Penicillin, streptomycin and methylthiazolyldiphenyl tetrazolium bromide (MTT) were procured from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Gefitinib powders and dimethyl sulfoxide (DMSO) were obtained from Aladdin Biotechnology (Shanghai, China). TGF-β1 and the cell cycle kit were purchased, respectively, from PeproTech (East Windsor, NJ, USA) and KeyGEN BioTECH (Nanjing, China). JAK2/STAT3 pathway inhibitor AG490 was obtained from MedChemExpress (Monmouth Junction, NJ, USA). For Western Blot analysis, primary antibodies against Cyclin D1, CDK4, p21, p53, E-cadherin, N-cadherin and vimentin were purchased from Proteintech Group Inc. (Chicago, IL, USA) and antibodies against snail, JAK2, STAT3, p-STAT3 and β-actin were obtained from Abcam Technology (Cambridge, UK). Goat antirabbit IgG and goat antimouse IgG were purchased from Abbkine (Wuhan, China).

After treatment, the cell slides were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes and then permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature. Normal goat serum was added to the slides and incubated for 30 minutes at room temperature. The primary antibody was then added and placed in a humid box overnight at 4°C. The next morning, the slides were washed 3 times with PBST and incubated with Dylight 549, Goat Anti-Mouse IgG(Abbkine, A23310) or Dylight 488, Goat Anti-Rabbit IgG (Abbkine, A23220) at 37°C for 1h, and the slides were stained with the blue DNA fluorescent stain 4’, 6-diamidino-2-phenylindole (DAPI) for 5 minutes. Finally, the slides were sealed with a mounting medium containing anti-fluorescence quencher, and observed under a fluorescence microscope.

Cells were homogenized in ice-cold RIPA lysis buffer containing protease inhibitor cocktail and phosSTOP (Roche, Basel, Switzerland). Equal amounts of protein (60 μg) were mixed with the loading buffer (Beyotime Institute of Biotechnology), boiled for 10 min, and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Temecula, CA). The membranes were blocked for 1 h in 5% milk. The membranes were then incubated overnight at 4°C with one of the following specific primary antibodies: rabbit anti-eNOS ser1177, anti-eNOS, anti-AMPKα thr172, anti-AMPKα, anti-β-actin (1 : 1 000, Cell Signaling Technology, Beverly, MA), anti-Akt ser473 (1 : 1 000, EPITOMICS, CA), anti-Akt (1 : 600, Proteintech), anti-TFAM (1 : 300, Proteintech), and anti-PGC-1α (1 : 200, Santa Cruz, CA). After washing, the membranes were incubated for 2 h at room temperature with secondary antibodies (Goat anti-rabbit IgG, goat anti-mouse IgG, 1 : 10 000, Abbkine, CA) and then washed. Finally, the blots were developed with enhanced chemiluminescence detection reagents (Thermo Scientific, Waltham, MA). Membranes were scanned using the MicroChemi bioimage analyzer (NDR, Israel) and quantified using Image J program and normalized against β-actin.

Total protein was extracted from frozen cells with radio-immunoprecipitation assay buffer. Extracted protein was quantified using a BCA assay kit. Protein samples were resolved by SDS–PAGE and then electro-blotted onto polyvinylidene fluoride membranes. The membrane was blocked and then incubated overnight at 4°C with the following primary antibodies: anti-TLR2 (1 : 1,000, ABCAM), anti-MyD88 (1 : 1,000, ABCAM), anti-NF-κB (1 : 1,000, ABCAM), or anti-Actin (1 : 10,000, Proteintech). On the next day, the membrane was subjected to incubation with goat antimouse IgG (1 : 10,000, ABBKINE) for 1 hr at RT. Immunoreactive proteins were visualized using enhanced chemiluminescence and analyzed with an image analyzer.

The primary antibodies used in this study were mouse anti-AAVR (Abcam, ab105385, 1:1,000), and mouse anti-β-actin (Abbkine, ABL1010, 1:2000). The secondary antibody used in this study was HRP conjugated Goat Anti-Mouse IgG (Abbkine, a21010, 1:4,000).
Cell debris were harvested, and cell lysates were prepared on ice using the RIPA lysis buffer (Beyotime, Shanghai, China) supplied with 1% inhibitor cocktail (Bimake, Shanghai, China). The lysates were centrifuged at 10,000 g for 5 min at 4°C and the supernatants were collected. Protein concentrations were quantified by the BCA assay (Thermo Fisher Scientific, Waltham, MA). Twenty μg of total protein was mixed with 6× SDS loading buffer (Sangon, Shanghai, China), followed by heating for 10 min at 95°C. The proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane (Biorad, Irvine, CA). The membrane was blocked in a blocking buffer (PBS with 5% nonfat milk and 0.1% Tween-20) for 1 h at room temperature. Then, the membrane was incubated with a primary antibody overnight at 4°C. The membrane was washed three times with PBST (PBS containing 0.1% Tween-20) and then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. The membrane was finally washed three times and developed using an ECL kit (Millipore, Temecula, CA).