Hif1a

Protein extracts were quantified by DC Protein assay (Bio-Rad), and equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis. After transfer of the proteins onto a nitrocellulose membrane (GE10600001, Sigma) for 1 hr at 100 mV, membranes were blocked in 5% milk, and specific proteins were labeled with the following antibodies: CPEB4 (Abcam Ab83009/clone 149C/D10, monoclonal homemade); HuR (3A2, sc-5261 Santa Cruz); HIF1a (Cayman 10006421); phospho-p44/42 (Erk1/2) (Thr202/Try204) (Cell Signaling clone E10, 9106); SOCS1 (Abcam Ab9870); phospho-p38 (Thr180/Y182) (Cell Signaling, 9211S); p38α (C-20)-G (Santa Cruz, sc-535-G); phospho-MAPKAPK2 (Thr222) (Cell Signaling, 3044S), TTP (D1I3T) (Cell Signaling, 71632), and vinculin (Abcam Ab18058). Quantification was done with ImageStudioLite software, and protein expression was normalized by the loading control signal.

Following intracardial perfusion of 4% paraformaldehyde, tissue was post-fixed for 1–2 h at 4 °C, cryoprotected in 30% sucrose, and embedded in OCT. Frozen sections were cut on a cryostat (20 μm) and stored at −80 °C. For staining, sections were thawed and then rehydrated in PBS. If necessary, tissues underwent antigen retrieval with citrate buffer (pH 6.0) at 95 °C for 10 min. Sections were blocked in 10% goat or donkey serum in 0.1% TritonX-100-containing PBS (blocking solution). Primary antibodies were diluted in blocking solution, and tissues incubated at 4 °C overnight or 2-h room temperature. For primary antibodies, we used PH3 (mouse monoclonal, Cell Signaling), Calbindin (mouse monoclonal or rabbit polyclonal, Swant), Cleaved Caspase 3 (rabbit polyclonal, Cell Signaling), NeuN (mouse monoclonal, Millipore), Iba1 (rabbit polyclonal, Wako), HIF1a (rabbit polyclonal, Cayman Chemicals), BNIP3 (rabbit polyclonal, Cell Signaling), and Cre (rabbit polyclonal, Millipore). Following primary incubation, tissues were washed with 0.1% Tween20-containing PBS, then incubated with proper Alexa Fluor secondary antibodies (Invitrogen) in blocking solution for 1 h at room temperature. Sections were mounted with fluoromount containing DAPI (Southern Biotech).