Anti gapdh antibody em1101

The protein isolation was performed as described previously [67 (link)]. The resulting proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA). The polyclonal anti-Flag A9044 (Sigma, St. Louis, MO) and monoclonal anti-GFP ab32146 (Abcam, Cambridge, UK) antibodies were used at a 1:5000 to 1:10 000 dilution for immunoblot analyses. The samples were also detected with monoclonal anti-GAPDH antibody EM1101 (Hangzhou HuaAn Biotechnology Co., Ltd.) as a reference. The intensity of immunoblot bands were quantified using the ImageQuantTL software.

The GFP, RFP, 3× Flag, or mCherry-fusion constructs were verified by DNA sequencing and transformed in pairs into PH-1. Transformants expressing pairs of fusion constructs were confirmed by western blot analysis. In addition, the transformants expressing a single fusion construct were used as references. For Co-IP assays, total proteins were extracted and incubated with the anti-GFP (ChromoTek, Martinsried, Germany) or anti-Flag (Abmart, Shanghai, China) agarose as described above. Proteins eluted from agarose were analyzed by western blot detection with a polyclonal anti-Flag A9044 (Sigma, St. Louis, MO), or an anit-GFP antibody (Abcam, Cambridge, UK). The protein samples were also detected with monoclonal anti-GAPDH antibody EM1101 (Hangzhou Huaan Biotechnology Co., Ltd.) as a reference. Each experiment was repeated twice.

For total protein extractions, mycelia were ground in liquid nitrogen. Approximately 200 mg of finely ground mycelia powder was resuspended in 1 mL of extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride [PMSF]) and 10 μL of protease inhibitor cocktail (Sangon, Shanghai, China). After homogenization with a vortex shaker, the lysate was centrifuged. The supernatant was mixed with protein loading buffer and boiled for 5 min. The resulting proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. To test the expression pattern of Nem1, Spo7, and Pah1 in complemented strains, monoclonal anti-GFP antibody 32146 (Abcam, Cambridge, MA, USA) was used at a 1:100,000 dilution for immunoblot analyses. The samples were also detected with monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody EM1101 (Hangzhou HuaAn Biotechnology Co., Ltd.) as a reference. Incubation with a secondary antibody and chemiluminescent detection were performed as described previously (52 (link)). Each experiment was repeated three times.