Hydroxylated hif 1α

Gastrocnemius tissue and HUVECs were lysed to extract proteins. Protein lysates (50 μg) were loaded and separated on the SDS‐PAGE gel. Then, proteins were transferred onto nitrocellulose membranes. Membranes were incubated with the corresponding primary antibodies against cleaved caspase‐3 (Abcam, ab49822), caspase‐3 (Abcam, ab13847), Bcl‐2 (Abcam, ab182858), BAX (Abcam, ab32503), LXRα (Abcam, ab176323), LXRβ (Abcam, ab28479), NOX4 (Abcam, ab109225), 3‐Nitrotyrosine (Abcam, ab52309), SIRT1 (Cell Signaling, #9475), hydroxylated HIF‐1α (Cell Signaling, #3434), FoxO1 (Cell Signaling, #2880), acetyl‐FoxO1 (Santa Cruz, sc‐49437), p53 (Cell Signaling, #2524), acetyl‐p53 (Cell Signaling, #2570) and β‐actin (Abcam, ab8227). Membranes were treated with an enhanced chemiluminescence kit (Millipore) and were imaged with UVP Bio‐Imaging Systems. QuantiOne imaging software (Bio‐Rad) was used for quantitative analysis by evaluating the integrated optical density.

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)].