Affinipure goat anti mouse igg h l

The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Tiangen) and cultured in a shaker of 37 °C and 180 rpm. When the OD₆₀₀ reached about 0.6, IPTG was added (final concentration 1 mmol/l; Sigma-Aldrich, St. Louis, MO, USA) to induce and express recombinant vimentin protein. The cell pellet was collected by centrifugation and lysed with sonication at 4 °C. Then the lysed product was analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the recombinant protein was present as a soluble protein or in inclusion bodies. The recombinant protein was purified according to the characteristics using a published gel purification method [18 (link)]. Purified recombinant vimentin protein (10 μg) was analyzed by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was blocked in 5% skim milk for 2 h at 37 °C and incubated with Anti-GST Tag Mouse Monoclonal Antibody (1:2000; CWBio, Beijing, China) for 2 h at 37 °C, and was then washed in phosphate-buffered saline (PBS) three times for 5 min and incubated with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (1:5000; Proteintech, Chicago, IL, USA) for 45 min at room temperature. Finally, imaging was obtained using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).

Cells were fixed with 4% paraformaldehyde in PBS for 8 min at room temperature, washed three times with 1X PBS and then blocked with 10% serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. Cells were again washed three times with 1X PBS and incubated a 1:200 dilution of rabbit polyclonal antibody against LC3B (Cat^# ab63817, Abcam) in PBS containing 0.1% Triton X-100 and 5% horse serum and overnight at 4 °C. After washing, the cells were incubated with fluorescein (FITC)-conjugated AffiniPure goat anti-mouse IgG (H + L) (Cat# SA00003-1, Proteintech) for 2 h at room temperature. Samples were mounted with Immu-mount (Thermo Scientific).
Cells were stained with 50 nM MitoTracker® Red CMXRos (Cat# 9082S, Cell Signaling Technology) for 30 min at 37 °C, washed with 1X PBS, and then used to detect the colocalization of LC3 and MitoTracker.

The cells were placed in cold RIPA solution (Solarbio, C0065) with PMSF (Solarbio, P0100) after washing the cells twice with phosphate buffer. Subsequently, they were pyrolysed with a protease inhibitor mixture for 30 min. The supernatant was centrifuged and harvested to analyse the protein concentration using the BCA Protein Assay Kit (BestBio, A045-4). Immunoblotting was conducted using the SDS-PAGE electrophoresis system. Briefly, 25 μL of sample was loaded and electrophoresed on a 10% SDS reducing gel. Blots were blotted onto a polyvinylidene fluoride membrane and incubated with primary antibodies against TLR3 (1 : 1000, Abcam, ab13915, USA), TLR4 (1 : 500, Abcam, ab13556), TLR6 (1 : 1000, Proteintech, 22240-1-AP), IDO1 (1 : 500, Abcam, ab55305), and NF-κB (1 : 500, Abcam, ab14059) overnight at 4°C. On the following day, the blot was washed three times with TBST and incubated with horseradish peroxidase-conjugated (HRP-conjugated) AffiniPure Goat anti-Mouse IgG (H+L) (1 : 5000, Proteintech, SA00001-1, USA) or HRP-conjugated AffiniPure Goat anti-Rabbit IgG (H+L) (1 : 5000, Proteintech, SA00001-2). Further, the blot was kept at 25°C for 1 h in TBST. Finally, proteins on the washed membrane were visualised using the TBST (Solarbio, T1082).

Two human urinary bladder cancer cell lines (T24 and UM-UC-3) that isolated from high-grade and late-stage tumors were used as MIBC models in this study [16 (link)]. T24 and UM-UC-3 were cultured in Dulbecco's Modified Eagle Medium (GIBCO-Invitrogen) containing 10% fetal bovine serum (ExCell Bio).The following antibodies were used in our study: Actin (Santa Cruz Biotechnology, sc-1616,1:1000), ZBTB7A (Santa Cruz Biotechnology, SC-33683, 1:1000), and HIC1 (Proteintech, 24949-1-AP, 1:1000), Mouse anti-Armenian hamster IgG-HRP (Santa Cruz Biotechnology, sc-2789, 1:2000) for ZBTB7A, HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2, 1:10000) for HIC1, HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1, 1:10000) for Actin. The primary antibodies were incubated for two hours at room temperature, while the secondary antibodies were incubated for one hour.

Cells were lysed in RIPA lysis buffer (Beyotime, P0013B) and PMSF (Beyotime, ST506). Cell lysis was carried-out for 30 min on ice, lysates were then centrifuged to remove insoluble material. Then, the loading buffer (Beyotime, P0015) was added to the lysates and heated at 100 °C for 3–5 min to denature the protein completely. Samples were separated on 12% SDS-PAGE Gel (Beyotime, P0053A), transferred to PVDF membranes, and detected with the indicated antibodies. The following antibodies were used for western blotting: anti-HPV18 E7 antibody (8E2, abcam, ab100953, dilution: 1:1000), anti-β-actin antibody (2D4H5, proteintech, 66009-1-Ig, dilution: 1:5000), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (proteintech, SA00001-1, dilution: 1:2000).

The RIPA buffer (1% Nonidet P40, 0.1% SDS, 0.5% deoxycholate, 50mM Tris (PH7.4) Protease Inhibitor Cocktail) for was used to extract proteins from colon tissue 15 mins. BSA standards were used to produce protein quantitative standard curves. The protein sample was placed in a boiling water bath for 6-10 mins for denaturation. 6%-8% SDS-PAGE gel was configured for protein electrophoresis. Then, membrane was rotated and immersed in the sealing liquid, and it was slowly sealed with a shaking table at room temperature for 1-2 h. Then the protein was transferred to PVDF membrane. The membrane was then cleaned twice by TBST. The membrane was incubated with diluted primary antibody and shaken overnight at 4° C. The first antibodies used were as follows: GAPDH (#60004-1-Ig, 1:8000, Proteintech, China), SRC (ab133283, 1:1000, Abcam, UK), P-SRC (#D7F2Q, 1:1000, Cell Signaling, USA), MAPK (#11257-1-AP, 1:2000, Proteintech), P-MAPK (#80031-1-RR, 1:10000, Proteintech), AKT1 (#60203-2-Ig, 1:10000, Proteintech) and P-AKT1 (#66444-1-Ig, 1:10000, Proteintech). The corresponding secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (#SA00001-1, Proteintech) was successively added and incubated in a shaking table for 1-2 h in dark at room temperature. The developer solution was added and then placed in a BIO-RAD for visualization.

Cells were rinsed twice with PBS, lysed in RIPA lysis buffer (Beyotime, China, CAT#P0013B) supplemented with a protease inhibitor (PMSF, Beyotime, China, CAT#ST506) and Phosphatase inhibitor cocktail A (Beyotime, China, CAT#P1082), and protein concentration was measured using the BCA reagent (Beyotime, China, CAT#P0012). Electrophoresis was conducted by 10% SDS-polyacrylamide gel and transferred proteins to PVDF membranes (Millipore, Cat# IPVH00010). The following primary antibodies were used for incubation overnight at 4 °C: 1:10,000 mouse anti-beta-Tubulin (Proteintech, China Cat#66240-1-Ig), 1:1000 rabbit anti-PKA (Cell Signaling, USA Cat#5842 T), 1:5000 rabbit anti-pCREB (Abcam, UK Cat# ab32096), and 1:1000 rabbit anti-CREB (Abcam, UK Cat# ab32515). All membranes were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, China Cat#SA00001-1) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, China Cat#SA00001-2) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) kit (Advansta, USA Cat#K-12045-D10) and quantified with a gel-image analyzing system (Fusion Optix, USA).