Anti phosphorylated erk1 2 thr180 tyr182

Femur explants were fixed in 4% paraformaldehyde and embedded in paraffin. Serial 5 μm sections were stained with hematoxylin–eosin-safran reagent using standard protocols. For immunohistochemical assessment, sections were labeled with the following antibodies and a Dako Envision Kit: anti-Col X (BIOCYC, N.2031501005; 1:50 dilution), anti-Sox9 (polyclonal antibody, Santa Cruz Biotechnology Inc., catalog D0609; dilution 1:75), anti-phosphorylated ERK1/2 (Thr180/Tyr182) (Cell Signaling Technology, #4370; 1:100 dilution), anti-phosphorylated p38 (Abcam, Ab4822; 1:200 dilution), and anti-Ki-67 (Abcam, Ab16667; 1:3 000 dilution). Images were captured with an Olympus PD70-IX2-UCB microscope and quantified using cellSens software.

After a 6-day culture period, fetal femur (E16.5) explants were fixed in 4% paraformaldehyde, decalcified with EDTA (0.4M), and embedded in paraffin. Serial 5 μm sections were stained with hematoxylin‑eosin‑safran (HES) reagent, using standard protocols. For immunohistochemical assessment, sections were labeled with the following antibodies and a Dako Envision Kit: anti-COLX (BIOCYC, catalog N.2031501005; 1:50 dilution), and anti–phosphorylated ERK1‑2 (Thr180/Tyr182) (Cell Signaling Technology, catalog 4370; 1:100 dilution). Images were captured with an Olympus PD70-IX2-UCB microscope and quantified using cellSens software.
Mean areas of individual hypertrophic chondrocytes were measured from COLX-labeled sections, within a 166 μm wide × 76 μm high box positioned 50 μm from mineralization front (Supplemental Figure 6). The measurements were made manually using Fiji software and the freehand selection tool. For analysis of the effect of the drug treatments on the area occupied by proliferative chondrocytes, these cells were identified by their round or columnar shape, as seen with HES staining, and by the absence of COLX labeling. We measured the total area occupied by chondrocytes within the whole growth plate and the area occupied by COLX⁺ chondrocytes. The area for proliferating chondrocytes was calculated by subtracting the COLX⁺ area from the whole growth plate area.

Whole-cell lysates and immunoprecipitated proteins were subjected to NuPAGE 4%–12% bis-tris acrylamide gel electrophoresis (Life Technologies). Using standard protocols, we probed the blots with the following primary antibodies: anti-phosphotyrosine (Cell Signaling Technology), anti-phosphorylated ERK1/2 (Thr180/Tyr182) (Cell Signaling Technology, #4370), anti-total ERK1/2 (Sigma-Aldrich, M5670), anti-phosphorylated p38 (Thr180/Tyr182) (Cell Signaling Technology, #9205), anti-total p38 (Cell Signaling Technology, #2708), anti-total PCNA (Abcam, ab29-100), and anti-β-actin (Millipore, MAB1501). Proteins were immunodetected with an anti-rabbit IRDye^® 800CW antibody (LI-COR Biosciences, 926-32211) and an anti-mouse IRDye^® 680RD antibody (LI-COR Biosciences, 926-68070). Immunoblots were quantified with ImageStudioLite software (LI-COR Biosciences).