Fusion sl4

CAV1 protein abundance was tested with tissue lysates from whole liver and isolated primary hepatocytes as previously described^{29 (link)}. Briefly, RIPA buffer with freshly added phosphatase and protease inhibitors was used to extract protein from tissue and cells. Protein concentration was assessed with the Bio-Rad protein assay kit according to the manufacturer’s instructions and quantified with the Tecan Infinite M200 via absorbance measurement at 690 nm. Equal amounts of protein (30 µg) were separated by SDS-PAGE (10–12% gels) and blotted onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat dry milk in TBST at room temperature for 1 h, and incubated overnight at 4 °C with primary antibodies. Primary antibodies against CAV1 (T3267, Cell Signaling) and GAPDH (sc25778, Santa Cruz Biotechnology) were used. HRP-linked anti-mouse (sc-2005, Santa Cruz) and anti-rabbit antibodies (sc-2357, Santa Cruz) were used as secondary antibodies. Signals were visualized by incubating the blots in Supersignal Ultra solution (Pierce, Hamburg, Germany) and recorded with imaging system Fusion SL4 (PEQLAB, Germany). The target protein bands were quantified by using Fluorchem Q system (ProteinSimple, California, USA).

For Western blot experiments, R. sphaeroides cultures were grown under semi-aerobic and ¹O₂ stress conditions as described above. Samples were taken at the indicated time points. Cells were broken by sonication and cell debris removed by centrifugation. Total protein extracts were separated on 10% PAA-SDS gels and transferred to nitrocellulose membranes (Whatman). Proteins were stained and fixed with Ponceau S (Sigma-Aldrich) and de-stained with sodium hydroxide. Membranes were blocked for 1 hour at room temperature with blocking buffer (1x TBS) containing 5% (w/v) milk powder (Roth). After blocking, the purified primary antibody α-GFP, diluted 1:4000 in blocking buffer, was added to the membrane and incubated for 1 hour. After washing the membrane 3 times for 5 min in 1x TBS buffer, the secondary antibody (anti-mouse IgG conjugated with horseradish peroxidase, produced in goat, Sigma-Aldrich) was added (diluted 1:5000 in blocking buffer) and the membrane further incubated for 1 hour at room temperature. The membrane was washed 3 times with 1x TBS for 5 minutes. Western blots were developed using the Lumi-Light Western Blotting Substrate 1 and 2 (Roche) and analyzed on a Chemiluminescence-Imaging System (Fusion-SL4; Peqlab).

For the SDS-PAGE the method previously described by Laemmli (Lassak et al., 2010 (link)) was used. Samples were taken from exponentially growing cultures and set to OD 10 in 2xSDS sample buffer. 10 μL of each sample was loaded onto a 12.5% SDS gel. FlhB-3xFLAG proteins were detected with Monoclonal ANTI_FLAG M2-Peroxidase mouse antibodies (Sigma–Aldrich), the protein membrane was treated with Western Lightning^® Plus-ECL, Enhanced Chemiluminescence Substrate Kit (Perkin Elmer, United States) and developed for 1 min using a Fusion SL4 (peqlab, Darmstadt).