Western lightning plus ecl chemiluminescence reagent

This technique was used for determination of protein expression of Nrf2, HO-1, pThr180/Tyr182-p38 MAPK, pThr183/Tyr185-JNK, Bax, and Bcl-2 in renal tissues [36 (link),37 (link)]. Briefly, the renal tissues were washed, homogenized (pre-cold lysis buffer), and supplemented with protease/phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). Total proteins determination was performed colorimetrically. Thirty µg of protein were incubated for 18–20 h at 4 °C with antibodies against Nrf2 (CAT # PA5-68817, Thermo Scientific, MA, USA), HO-1 (CAT.# ab13243, Abcam, CB, UK), JNK (CAT.# ab179461, Abcam, CB, UK), p-p38MAPK (CAT.# b170099, Abcam, CB, UK), Bax (CAT # MA5-14003, Thermo Scientific, MA, USA), Bcl-2 (CAT# PA5-27094, Thermo Scientific, Waltham, MA, USA), and β-actin (1:2500, # A5060, Sigma, St. Louis, MO, USA). After membrane washing, suitable secondary antibodies (Dako, Glostrup, Denmark) incubation was done. The Western Lightning Plus ECL Chemiluminescence Reagents (Perkin Elmer, Waltham, MA, USA) were mixed, applied, and the band signals/intensities were then captured via Chemi-Doc imager (Bio-Rad, Hercules, CA, USA).

Antibodies against R. rubrum BacA were raised by immunization of rabbits with purified BacA-His₆ protein (Eurogentec, Belgium). Cells were harvested in the exponential growth phase. Immunoblot analysis was conducted as described previously (Thanbichler and Shapiro, 2006 (link)), using anti-BacA antiserum (1:10.000), a monoclonal anti-mNeonGreen antibody (Chromotek, Germany; Cat. #: 32f6; RRID: AB_2827566), a polyclonal anti-GFP antibody (Sigma, Germany; Cat. #: G1544; RRID: AB_439690), a polyclonal anti-mCherry antibody (BioVision, USA; Cat. #: 5993; RRID: AB_1975001) or a monoclonal anti-HA antibody (Merck Millipore, USA; Cat. #: 05–904; RRID: AB_11213751) at dilutions of 1:10,000, 1:1000, 1:10,000, 1:10,000 or 1:1000, respectively. Goat anti-rabbit immunoglobulin G conjugated with horse-radish peroxidase (Perkin Elmer, USA) or goat anti-mouse immunoglobulin G conjugated with horse-radish peroxidase (Sigma, Germany) were used as secondary antibodies. Immunocomplexes were detected with the Western Lightning Plus-ECL chemiluminescence reagent (Perkin Elmer, USA). The signals were recorded with a ChemiDoc MP imaging system (BioRad, Germany) and analyzed using Image Lab software (BioRad, Germany).