Pierce fast western blot kit

Protein samples were separated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA) for protein bands detection. The membranes were blocked in 5% nonfat milk. Primary antibodies including anti-GAPDH (1:2000, Abmart, Shanghai, CHN), anti-BCL2 (1:500, Abcam, Cambridge, UK), anti-Caspase 9 (1:1000, Cell Signaling Techonolgy, MA, USA), anti-cleaved-Caspase 9 (1:500, Cell Signaling Techonolgy, MA, USA), anti-Caspase 3 (1:1000, Abcam, Cambridge, UK), and anti-cleaved-Caspase 3 (1:1000, Abcam, Cambridge, UK) were used for incubation overnight at 4 °C. The membranes were incubated with HRP-conjugated secondary antibody (1:3000, Jackson Immuno Research, PA, USA) at room temperature for 1 h and the protein bands were detected by Pierce Fast Western Blot Kit (Thermo Fisher Scientific, MA, USA).

The method of extracting total protein from cultured cells and specimens was to use RIPA buffer (Applygen, China) according to the manufacturer’s instructions. SDS-PAGE and Western blot were performed according to standard protocols. Immunoreactive bands were visualized by Pierce Fast Western Blot Kit (ThermoFisher, USA) using an SYNGENE G: BOX imaging system (Frederick).
For immunofluorescence, we seeded stable RCC cells in 24-well plates. 4% formaldehyde was used to fix the cells (~70–90% confluency) for 15 min at room temperature. PBS wash three times for 5 min each. Block specimen in Blocking Buffer for 60 min. Aspirate blocking solution, apply diluted primary antibody. Incubate overnight at 4 °C. Rinse three times in PBS for 5 min each. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 h at room temperature in the dark. Rinse three times in PBS for 5 min each. Coverslip slides with DAPI for 5 min. The images were captured by a Confocal microscope (Leica, Heidelberg, Germany). All of the above solutions were included in Immunofluorescence Application Solutions Kit (Cell signaling technology, #12727, USA). The antibody conjugated to the primary antibody, and the secondary HRP is described in Supplementary Table 4.

To confirm equal expression levels of the BiFC constructs, cell lysates were evaluated using Immunoblot analysis. In brief, the cells of an overnight culture of the different constructs were spun down (5.500 g, 5 min.) and diluted in PBS to an OD₆₀₀ of 5. Each sample was resuspended in 200 µl 1 × SDS resuspension buffer (46 μl 4 × Laemmli sample buffer (BioRad), 20 mM DTT, 150 μl mQ H₂O) and heated for 8 min at 95° C. Cell debris were spun down (15.000 g, 5 min) and 14 µl of each sample were loaded on a SDS-PAGE gel (BioRad Protean TGX, 4–20%, 16 min, 300 V). A fast blotting procedure was performed (BioRad Trans-Blot Turbo, TGX Turbo protocol) to a PVDF membrane (Amersham Hybond PVDF). Three different antibodies were used in separate immunoblots directed towards the YFP split YFP variants (anti YFP_C: Roche Diagnostics #11814460001; anti YFP_N: BioLegend #902601), respectively, using a Pierce Fast Western Blot kit (ECL substrate) from Thermo Scientific. The chemiluminescence signal was detected via luminol enhancement from the kit and detection was manually performed and captured using a ChemiDoc MP imager from BioRad and Image Lab software.

Proteins were harvested from cells and colon tissues with RIPA Lysis Buffer (Beyotime, Hainan, Jiangsu, China) supplemented with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor and phosphatase inhibitor. Total protein concentration were determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, Massachusetts, USA), denatured protein samples of appropriate quality of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. Then membranes were later blocked with 5% skimmed milk, and incubated were immunodetected with specific antibodies against occludin ( 71–1500, Thermofisher, CA, USA), ZO-1 (ARG55738, Arigo, Taiwan, China), IL-1β (12242; Cell signaling Technology, Massachusetts, USA), TNF-α (a11534, Abclonal, Wuhan, China), and GAPDH (Antgene, Wuhan, China) overnight at 4 °C. The secondary antibody was purchased from GeneTex (Irvine, California, USA). Protein bands were visualized by the FluorChem Imaging System (ProteinSimple, San Jose, California, USA) using the commercial Pierce™ Fast Western Blot Kit and the ECL Substrate (Thermo Fisher, Waltham, Massachusetts, USA).

Cell lysates were subject to SDS–PAGE on 4–20% Bis‐Tris gels and transferred to PVDF membranes. Pierce Fast Western Blot Kit (Thermo Scientific) was used to perform immunoblot according to the manufacturer's instructions with the following antibodies: mouse anti‐His‐tagged (Millipore 05‐949) and HRP‐conjugated mouse anti‐tubulin (Proteintech HRP‐66031). Chemiluminescence of anti‐His‐tagged antibody was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).