Horseradish peroxidase hrp conjugated anti mouse igg

Yeast cells were grown to logarithmic phase in YPGA medium at 28 °C under different light conditions. Protein extraction from yeast cells was performed as described⁶² . The extracted proteins (40 μg) were separated in a 4–15% SDS-polyacrylamide gel [Bio-Rad Laboratories, Hercules, CA] and blotted onto polyvinylidene difluoride (PVDF) membrane [Bio-Rad], and immunoblotting was performed using mouse anti-HA monoclonal antibody [MBL, Nagoya, Japan] and horseradish peroxidase (HRP)-conjugated anti-mouse IgG [Promega, Madison, WI]. The signals were detected using an LAS-3000 imaging system [FUJIFILM, Tokyo, Japan]. The raw image is presented in Supplementary Fig. S5, which includes an unprocessed original data that was used to prepare Fig. 1d.

Full-length RGLG3 and RGLG4 were amplified by PCR, and each was cloned into pRTL-GFP, generating a construct expressing an N-terminal GFP fusion protein. Arabidopsis mesophyll protoplast preparation, transfection and 4,6-diamidino-2-phenylindole (DAPI, 1mg/ml; Sigma, USA) staining were performed as described (He et al., 2011b (link)). Confocal imaging was performed with a Zeiss LSM 710 NLO laser scanning confocal microscope (excitation 488nm; emission 505–550nm). Expression of GFP alone (from the control pRTL-GFP), GFP-RGLG3 and GFP-RGLG4 was confirmed by western blotting using anti-GFP (Abcam, Germany) as the primary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Promega, USA) as the secondary antibody.