Arg55258

Total protein was obtained from snap-frozen hearts or cells using a protein extraction kit (Keygenbio, KGP250) and a 1.5 mL tube (Guangzhou Jet Bio-Filtration Co., Ltd.). Thirty micrograms of protein was separated by SDS–PAGE and then transferred onto PVDF membranes. The membranes were incubated at 4°C overnight with conjugated antibodies (Sino Biological Inc., 1 : 2000). The immunoblotting bands were detected by chemiluminescence. The following antibodies were used: anti-VEGF-C (22601-1-AP, Proteintech), anti-VEGFR-3 (ab273148, Abcam), anti-p-p38 (ARG51850, Arigo), anti-p38 (ARG55258, Arigo), anti-VE-cadherin (ab205336, Abcam), anti-p-ERK1/2 (4370, CST), anti-ERK1/2 (4695S, CST), anti-p-AKT (9271S, CST), anti-AKT (9272, CST), anticalcineurin A (CaNA) (2614, CST), anti-p-STAT3 (9145, CST), anti-STAT3 (9139, CST), anti-TGF-β1 (3711, CST), anti-p-Smad2/3 (882S, CST), anti-Smad2/3 (8685, CST), anti-NOX2 (19013-1-AP, Proteintech), anti-NOX4 (14347-1-AP, Proteintech), anti-p-P65 (3033, CST), anti-P65 (4764, CST), anti-β2i (13635, CST), anti-β5i (ARG41022, Arigo) anti-MKP5 (374276, Santa), and anti-GAPDH (5174, CST). ImageJ software was used for densitometry analysis.

Primary cells were extracted from the flat bone of P3 Ddr1^f/f-4OHT and OKOΔDdr1 mice and MC3T3-E1 cells were extracted with RIPA extraction buffer (89900, Thermo Fisher Scientific Inc., Waltham, MA, USA.) containing 1% proteinase inhibitor (78429, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% phosphatase inhibitor (78426, Thermo Fisher Scientific Inc., Waltham, MA, USA). Western blot was performed with primary antibodies against DDR1 (ab22719, Abcam, Cambridge, MA, USA), collagen type I (ab34710, Abcam), collagen type X (ab58632, Abcam), GAPDH (MA5-15738, Thermo Fisher Scientific Inc.), ALP (ab95462, Abcam, Cambridge, MA, USA), Runx2 (ab23981, Abcam), BMP2 (ab14933, Abcam), Col-I (ab34710, Abcam), Osteocalcin (GTX13418, Gene Tex Inc., Irvine, CA, USA), phosphorylated 44/42 ERK1/2 (D11A8, Cell Signaling Inc., Danvers, MA, USA), ERK1/2 (137F5, Cell Signaling Inc.), phosphorylated p38 (Thr180/Tyr182) (ARG51580; Arigo biolaboratories, Hsinchu, TW), p38 (ARG55258; Arigo biolaboratories) and mouse and rabbit secondary antibodies (615005214 and 611005215; Jackson immune research; West Grove, PA, USA). Protein bands were detected by enhanced chemiluminescence analysis (ECL system; GE Healthcare, Piscataway, NJ, USA).