psenp2 gfp  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: SENP2 Promotes VSMC Phenotypic Switching via Myocardin De-SUMOylation

doi: 10.3390/ijms232012637

Figure Lengend Snippet: Myocardin is SUMOylated at lysine 573, which can be de-SUMOylated by SENP2. ( A ) HEK-293T cells were transfected with Flag-myocardin/WT (Flag-MYOCD/WT) or Flag-myocardin/K573R (Flag-MYOCD/K573R), HA-SUMO1, and Ubc-9 for 24 h. The SUMOylation of Flag-MYOCD was determined by the IP assay using Flag beads and Western blotting using anti-Flag, anti-HA, and anti-GAPDH antibodies. ( B ) HEK-293T cells were transfected with Flag-myocardin (Flag-MYOCD), HA-SUMO1, RGS-SENP2, and Ubc-9 for 24 h. The SUMOylation of Flag-MYOCD was determined by the IP assay using Flag beads and Western blotting using anti-Flag, anti-HA, and anti-β-actin antibodies.

Article Snippet: Adenovirus containing pADV-mCMV-SENP2-3xFlag-P2A-EGFP (GFP-SENP2) and pADV-mCMV-3xFlag-P2A-EGFP (GFP-Con) was purchased from OBiO technology (Shanghai, China).

Techniques: Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SENP2 Promotes VSMC Phenotypic Switching via Myocardin De-SUMOylation

doi: 10.3390/ijms232012637

Figure Lengend Snippet: SENP2 promotes proteasome-dependent degradation of myocardin. ( A ) HEK-293T cells were transfected with Flag-myocardin (Flag-MYOCD) and increasing amounts of Flag-SENP1, Flag-SENP2, or Flag-SENP3 for 24 h. The levels of Flag-MYOCD in whole-cell lysates were determined by Western blotting with anti-Flag and anti-α-tubulin antibodies (n = 3, * p < 0.05). ( B ) HEK-293T cells were transfected with Flag-MYOCD, HA-SUMO1, and Flag-SENP1, Flag-SENP2, or Flag-SENP3 for 24 h. Lysates were prepared and analyzed by Western blotting (n = 3, * p < 0.05 vs. Lane 1, # p < 0.05 vs. Lane 2). ( C ) HEK-293T cells were transfected with Flag-MYOCD and increasing amounts of RGS-SENP2 for 24 h, in the presence or absence of MG132 (10 μM) for the last 10 h. Lysates were prepared and analyzed by Western blotting (n = 3, * p < 0.05).

Article Snippet: Adenovirus containing pADV-mCMV-SENP2-3xFlag-P2A-EGFP (GFP-SENP2) and pADV-mCMV-3xFlag-P2A-EGFP (GFP-Con) was purchased from OBiO technology (Shanghai, China).

Techniques: Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SENP2 Promotes VSMC Phenotypic Switching via Myocardin De-SUMOylation

doi: 10.3390/ijms232012637

Figure Lengend Snippet: SENP2 promotes VSMC phenotypic switching in vitro. ( A ) VSMCs were infected with control lentivirus (Sh-Con) and sh-SENP2 lentivirus (Sh-SENP2) for 72 h. The expression of SENP2, α-SMA, SM22α, and SM-MHC was examined by real-time quantitative PCR (RT-qPCR). ( B ) VSMCs were infected with adenovirus containing GFP-Con or GFP-SENP2 for 48 h. The expression of SENP2 was examined by Western blotting. The expression of α-SMA, SM22α, and SM-MHC was examined by RT-qPCR. ( C ) VSMCs were infected with control lentivirus (Sh-Con) and sh-SENP2 lentivirus (Sh-SENP2) for 48 h. Monolayer confluent cells were serum-starved overnight and scraped in the presence of normal growth medium to stimulate VSMC migration toward the wound area. Representative images of the in vitro scratch-wound assay and quantification of migrated cells are presented. Scale bars: 200 µm. ( A – C ), n = 3, * p < 0.05.

Article Snippet: Adenovirus containing pADV-mCMV-SENP2-3xFlag-P2A-EGFP (GFP-SENP2) and pADV-mCMV-3xFlag-P2A-EGFP (GFP-Con) was purchased from OBiO technology (Shanghai, China).

Techniques: In Vitro, Infection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Migration, Scratch Wound Assay Assay

Journal: International Journal of Molecular Sciences

Article Title: SENP2 Promotes VSMC Phenotypic Switching via Myocardin De-SUMOylation

doi: 10.3390/ijms232012637

Figure Lengend Snippet: Schematic illustrating how SENP2 promotes VSMC phenotypic switching. In this study, we propose that SENP2 promotes VSMC phenotypic switching via de-SUMOylation of myocardin and regulation of its protein stability.

Article Snippet: Adenovirus containing pADV-mCMV-SENP2-3xFlag-P2A-EGFP (GFP-SENP2) and pADV-mCMV-3xFlag-P2A-EGFP (GFP-Con) was purchased from OBiO technology (Shanghai, China).

Techniques: