Elongin c

Wogonin was isolated from S. baicalensis Georgi according to the protocols reported previously with slight modifications [21 (link)]. Wogonin was dissolved in dimethylsulfoxide (DMSO) as a stock solution and stored at −20°C. Further dilution was performed in culture medium prior to each experiment (DMSO%<0.1%). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Fluka and dissolved in 0.01 M PBS. The enzyme-linked immunosorbent assay (ELISA) kit for VEGF was purchased from BosterBio (Pleasanton, USA). The rabbit polyclonal antibodies to c-Myc, HIF-1α, VEGF, VHL, CUL2, Flag-tag and PIASy were purchased from Bioworld. The mouse polyclonal antibody to β-actin was obtained from Boster. Other antibodies to Rbx1, Elongin C, Elongin B and HA were from Santa Cruz Biotechnology. IRDyeTM 800 conjugated secondary antibodies were obtained from Rockland Inc. Five-to-six-week-old female BALB/c-nude mice (SLACAS, Shanghai, China) were used for xenograft assays. The mice were fed ad libitum throughout the experimental period.

Proteins were obtained as previously described (28 ) and resolved on a 12.5% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to polyvinylidene fluoride (PVDF) membranes, blots were blocked and probed with different primary antibodies: VHL (BD Biosciences, # 556347, diluted 1/5,000), GFP (Santa Cruz, sc-8334, diluted 1/1,000), HIF-2α (Novus Biologicals, NB100-122, diluted 1/1,000), β-actin (Cell Signaling, #4970, diluted 1/1,000), Elongin B (Santa Cruz, sc-133090, diluted 1/500), and Elongin C (Santa Cruz, sc-1559, diluted 1/500). The following secondary antibodies were used accordingly: anti-rabbit (Cell Signaling, #7074, diluted 1/5,000), anti-goat (Santa Cruz, sc 2020, diluted 1/2,000), and anti-mouse (Cell Signaling, #7076, diluted 1/2,000).

Cells were lysed in passive-lysis buffer (Promega) followed by gentle sonication. Cell lysates were incubated with a suitable antibody in the presence of protein A sepharose beads (Amersham). In the hydroxylated-HIF-1α peptide competition assay, biotinylated wild-type or proline hydroxylated-peptides (corresponding to HIF-1α residues 556–574) were synthesized (American Peptide Company) and then dissolved in sterile water (500 μg/ml). The peptide was subsequently added to the immunoprecipitation mixture, after which the following antibodies were used for immunoblot and immunoprecipitation: HIF-1α, α-tubulin, GFP, HA, myc epitope, PHD2, Elongin C and GST (Santa Cruz Biotechnology); hydroxylated-HIF-1α (Cell Signaling Technologies); FLAG (Sigma); VHL (BD Bioscience). A polyclonal anti-PLD antibody that recognizes both PLD1 and PLD2 was generated as previously described [36 (link)].