Anti flag a9044

For western blotting assay, protein samples of strains were prepared and extracted as described previously [96 (link)]. Proteins separated on the SDS-PAGE gel were transferred onto a polyvinylidene fluoride membrane with a Bio-Rad electroblotting apparatus. The polyclonal anti-Flag A9044 (Sigma, St. Louis, MO, USA), monoclonal anti-GFP ab32146 (Abcam, Cambridge, MA, USA) and monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibodies were used at a 1:2000 to 1:10000 dilution for immunoblot assays. The samples were also detected with the monoclonal 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 [97 (link)]. The experiment was repeated three times independently.

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.

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.