Anti g6pdh antibody

Western blotting assays were carried out by standard techniques. Anti-G6PDH antibody (Sigma; 1:50000 dilution) was used as loading control. Monoclonal anti-HA antibodies (Clone 12CA5, Roche; 1:1000) were used as primary antibodies to detect the expression of the HA-tagged regulatory PI3K subunits.

Total protein was extracted by using an NaOH-lysis method for measuring Sch9 levels [111 (link)]. About 1×10⁸ cells were resuspended in 1 ml lysis buffer (0.185 M NaOH, 0.75% β-ME) and kept on ice for 10 minutes. Trichloroacetic acid (TCA) was added to the lysis buffer at a final percentage of 8% to concentrate proteins for electrophoresis. After 15 minutes, all residual supernatant was removed and the precipitant was resuspended in HU sample buffer (8 M Urea, 5% SDS, 0.2 M Tris-HCl pH 6.5, 1 m M EDTA, 0.01% bromophenol blue) supplemented with 2 M Tris base in a 50:3 ratio. The protein lysate was then incubated at 65°C for 10 minutes. Total protein lysate was separated by 6% SDS-PAGE, and blotted onto PVDF membrane (Immobilon-PSQ, Millipore, Billerica, MA) in a transfer buffer (25 mM Tris base, 200 mM glycine, 20% methanol). Membranes were blocked in 1% casein (Sigma C5890) blocking solution. Mouse monoclonal anti-hemagglutinin (anti-HA) antibody (Covance, Princeton, NJ) was used to detect HA-tagged proteins. G6PDH was used as an internal control protein and was detected by rabbit polyclonal anti-G6PDH antibody (Sigma A9521). For the HA-tagged construct, at least three independent transformants from each strain were tested to ensure that consistent patterns were observed between clones.

Techniques used for obtaining yeast extracts, fractionation by SDS-PAGE, and transfer to nitrocellulose membranes have been previously described [67 (link)].
Immunodetection was carried out with the following primary antibodies: rabbit polyclonal anti-phospho-p44/42 (Thr202/Tyr204) MAPK antibody (Cell Signaling, Danvers, MA, USA) for dually phosphorylated Slt2, mouse monoclonal anti-Slt2 (E-9) antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for total Slt2 protein [67 (link)], mouse monoclonal anti-Myc (4A6) antibody (Merck, Darmstadt, Germany,) for Myc-tagged Rlm1, rabbit polyclonal anti-phospho-Ypk1 (T662) (1:20,000), kindly provided by Dr. Ted Powers, for phospho-T662-Ypk1, mouse monoclonal anti-HA 12CA5 (1:1000, Sigma-Aldrich, St. Louis, MO, USA) for HA-tagged Ypk1, and rabbit polyclonal anti-G6PDH antibody (Sigma-Aldrich, He) for G6PDH recognition (loading control). Fluorescent-conjugated secondary antibodies were used to detect primary antibodies with an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).