Anti rabbit igg secondary antibody

To assess LC3-I/II, p62/SQSTM1, HSPA5 and GAPDH expression, western blot analysis was carried out on 10% or 15% SDS-PAGE as previously described [38 (link)]. The anti-LC3 and anti-GAPDH antibodies were purchased from Medical & Biological Laboratories (MBL, Japan). The anti-HSPA5 antibody was purchased from Abcam (Cambridge, UK). The anti-p62/SQSTM1 antibody was purchased from Abways Technology (Shanghai, China). Anti-rabbit IgG secondary antibody and anti-mouse IgG secondary antibody were purchased from Cell Signaling (Danvers, USA). The dilution ratio of all antibodies was 1:1000. Band intensities were quantified using ImageJ software (NIH, USA).

CORM-3 was purchased from Sigma Japan Inc. (Tokyo, Japan). CORM-3 solution was prepared by dissolving the compound in distilled water when it is in need. Meanwhile, iCORM-3 is prepared by dissolving CORM-3 in a phosphate buffer and standing to release CO at room temperature for 48 hours. Eliminate residual CO by bubbling the previous solution with N₂ [20 (link)]. The following reagents were purchased from designated sources: anti-E-cadherin rabbit polyclonal antibody (cat no. 20874-1-AP), anti-N-cadherin rabbit polyclonal antibody (cat no. 22018-1-AP), anti-Keap-1 rabbit polyclonal antibody (cat no. 10503-2-AP), and anti-Nrf2 rabbit polyclonal antibody (cat no. 16396-1-AP) were purchased from Proteintech Group, Inc. (Chicago, IL, USA). Anti-HO-1 rabbit polyclonal antibody (cat no. ab13243) was the product of Abcam (Cambridge, MA, USA). Anti-GAPDH rabbit monoclonal antibody, anti-rabbit IgG secondary antibody, and anti-Ki67 rabbit monoclonal antibody were from Cell Signaling Technology (Beverly, USA).

Western blotting was performed using a previously described protocol [31 (link)]. The seeded cells were removed from the culture dishes and centrifuged, and the resulting cell pellets were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). The lysates were mixed with sample buffer (Sigma, St. Louis, MO, USA) at a 1:1 ratio and then resolved on SDS–PAGE gels. After electrophoresis, the proteins were transferred to a PVDF membrane; the membrane was incubated with the primary antibody against GDF15 (1:1000, rabbit monoclonal antibody, no. 8479; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight, anti-rabbit IgG secondary antibody (1:1000, no. 7074; Cell Signaling Technology) for one hour at room temperature, and an anti-β-actin polyclonal antibody (MBL, Tokyo, Japan) for one hour at room temperature. Blots were visualized using an Amersham Imager 600 (Cytiva, Tokyo, Japan) and Immobilon Clasico (Sigma–Aldrich, St. Louis, MO, USA). The immunoblots were evaluated by quantifying the band intensity using ImageJ software.

Culture supernatants were subjected to 12% SDS-PAGE and electrophorectically transferred to 0.45 μm pore size PVDF membrane (Millipore Inc., Burlington, CA, United States). The membrane was blocked in 5% (w/v) skim milk in TBST buffer (0.3% Tris, 0.8% NaCl, 0.02% KCl, 0.1% Tween 20) for 1 h and then incubated with affinity purified polyclonal rabbit anti-PG-1 antibody at 1:500 dilution, synthesized from Biomatik (Cambridge, ON, Canada) overnight at 4°C. After washing the membrane three times at 5 min each in TBST, the membrane was incubated with anti-rabbit IgG secondary antibody (1:1000 dilution) conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA, United States) for 1 h at room temperature. Bands were visualized using an ECL Plus Western blotting system according to manufacturer’s instruction (Amersham Biosciences, Piscataway, NJ, United States).

Western blot analysis was carried out using an iBind Flex system (ThermoFisher Scientific) for primary and secondary antibody immunoblotting following a previously described method.^{29 (link)} For cell lysate collection SKOV3-ip1 and SKOV3-TRip2 cells were seeded in 6-well plates for 24 h at a density of 1.25 × 10⁵ mL⁻¹ and 1.75 × 10⁵ mL⁻¹, respectively, in 2 mL of media. Lysates were collected in RIPA buffer, protein quantified by Pierce BCA assay and 20–30 μg loaded on a 10% polyacrylamide gel which was run at 200 V. Rabbit monoclonal primary antibodies against anti-ALDH1A1 (Cell Signaling Technology) was used at a concentration of 1 : 1000 and anti-rabbit IgG secondary antibody (1 : 200 dilution; Cell Signaling Technology) for cell lysates analysis. Actin was used as a loading control for all experiments (1 : 1000 dilution; Cell Signaling Technology). After antibody incubations membranes were washed 5× with tris-buffered saline with tween (TBST) and visualised using ECL regent (GE Healthcare), with images taken using an iBright CCD camera (Invitrogen). Images were always acquired within the linear range of the camera to prevent the overexposure of any blots.

Cells were solubilized with a cell lysis buffer (Cell Signaling Technology, Danvers, MA), 1 mM phenylmethylsulfonyl fluoride (PMSF; Cell Signaling Technology), and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology) for 30 min at 4°C. Cell lysates were centrifuged at 14,000 ×g for 20 min at 4°C, and the supernatant was used for Western blotting. After quantification of protein concentration each sample, the samples (50 μg of total protein per lane) were run on 12% SDS-PAGE gels (BioRad, Hercules, CA) and immunoprobed with COX-2 and BCL2A1 protein. Results were visualized by SuperSignal^™ West Femto chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA). Rabbit monoclonal antibodies for COX-2 (Cell Signaling Technology) and BCL2A1 (Abcam, Cambridge, UK) were used at 1:1,000 and 1:500 dilutions, and anti-rabbit IgG secondary antibody was used at 1:20,000 dilution (Cell Signaling Technology). Antibody against β-actin (Cell Signaling Technology) at a dilution of 1:1,000 was used as a loading control. Signals were detected with an Amersham Imager 600 (GE Healthcare).