Fig. 2: GmBTB/POZ promotes the ubiquitination and degradation of GmLHP1.

a GmLHP1 protein was degraded, which likely occurred primarily through the 26S proteasome. For MG132 treatment, WT soybean plant extracts were treated with 100 µM MG132 for 1 h and incubated with GmLHP1-His protein for the indicated time. GmActin was used as a loading control. b Relative band density of GmLHP1-His. GmLHP1-His was quantified using ImageJ software. c–i In vitro cell-free degradation assays of GmLHP1-His in protein extracts from GmBTB/POZ transgenic soybean plants. Protein extracts from transgenic (GmBTB/POZ-OE and GmBTB/POZ-RNAi) and WT soybean plants were incubated with GmLHP1-His for the indicated time. GmLHP1-His levels were visualized by immunoblotting using anti-His antibody. GmActin was used as a loading control. The protein level of 0 h was set to 1.00. j Diagram of the plant binary expression vector system (p35S: Flag-GmLHP1 + p35S: GmBTB/POZ-Myc). k GmBTB/POZ promotes the ubiquitination of GmLHP1 in vivo. GmLHP1-Flag was immunoprecipitated using anti-Flag-Tag Mouse mAb (Agarose Conjugated) from GmLHP1-OE and GmLHP1-OE/GmBTB/POZ-OE transgenic soybean hairy roots by high-efficiency A. rhizogenes-mediated transformation. The transgenic hairy roots were treated with 100 µM MG132 for 8 h before extraction. The immunoprecipitated protein was examined using anti-Flag and anti-ubi antibodies.