P erk 4370

GPR30 (ab37742), vimentin (ab92547), cardiac troponin I (ab47003), TGF-β1 (ab27969), and MMP-9 38,898) were purchased from Abcam. ERK 4,695) and p-ERK (4,370) were purchased from Cell Signaling Technology. GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States ). The rabbit anti-goat, goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China). The MMP-9 ELISA kit was purchased from Elabscience Bioengineering Institute (Wuhan, China). GPR30 agonist G1 (10,008,933) and GPR30 antagonist G15 14,673) were purchased from Cayman Chemical (Ann Arbor, MI, United States ). The ERK inhibitor PD98059 (HY-12028) and DOX (HY-15142) were purchased from MCE. AAV-GPR30 was purchased from the GeneChem Company (Shanghai, China). DAPI was obtained from Roche Molecular Biochemicals (Bayer, Mannheim, Germany). The ECL reagent was purchased from Millipore (Billerica, MA, United States ).

Cells were plated in 6 well plates 24h prior to protein extraction. Cells were washed with phosphate buffered saline (PBS) and lysed using radio-immunoprecipitation (RIPA) buffer [150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS] in 50 mM Tris·HCl (pH 8.0) supplemented with 1x protease and phosphatase inhibitors (78442; Pierce). Protein concentrations were determined using the BCA Protein Assay kit (23235; Pierce). Total protein in 1×Laemmli buffer with 10% 2-mercaptoethanol was separated by SDS/PAGE, transferred for 1 h to a PVDF membrane (IPVH00010; Millipore) by electro blotting with 20% (vol/vol) methanol, and blocked for 1 h in 5% (wt/vol) dry milk/Tris-buffered saline (TBS)/0.1% (vol/vol) Tween-20. Membranes were incubated overnight at 4°C with primary antibodies following incubation with horseradish peroxidase-conjugated secondary antiserum for 1 h and developed using enhanced chemiluminescence [32106 (Pierce) or 64 – 201BP (Millipore)]. β –Actin protein expression served as a loading control. P-ERK (4370), p-AKT (4060), and p-S6 (4857) were obtained from Cell Signaling Technology, β –Actin was purchased from Sigma Aldrich.

Microglia were labelled using a rabbit primary antibody targeting ionized calcium binding adapter molecule 1 (IBA‐1) (019‐19741) from Wako (Richmond, VA, USA). Antibodies against basigin (Rabbit polyclonal antibody used for Western blot, ab64616; Rat monoclonal antibody used for immunofluorescence double labelling, ab34016), PECAM (ab9498), IGF‐1 (ab9572), IGF‐1 receptor (ab16890), VEGF (ab51745) and VEGFR‐2 (ab5473) were purchased from Abcam (Cambridge, MA, USA). HIF‐1α (MAB 5382) was obtained from Millipore (Billerica, MA, USA). Antibodies against AKT (9272), P‐AKT (4060), ERK (4695), P‐ERK (4370) and the MEK1/2 inhibitors PD98059, PI3K inhibitor LY294002 were from Cell Signaling (Andover, MA, USA). Antibodies against β‐actin were from Santa Cruz (Santa Cruz, CA, USA). Recombinant human IGF‐1 (100‐11) was from ProteinTech (Rochy Hill, NJ, USA). Secondary antibodies including goat anti‐rabbit conjugated to AlexaFluor 594/CY3 or AlexaFluor 488/FITC were purchased from Beijing ComWin (Beijing, China). SYBR Premix Ex Taq II and Multiscript RT were purchased from TaKaRa (Dalian, China).

Cells were lysed in RIPA lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 50 mM EDTA) with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific). Lysates were run on SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-ICAM-1 BBIG-I1 (1:1,000), VCAM-1 BBIG-V1 (1:1,000), E-selectin BBIG-E4 (1:1,000) (R&D systems), MAP4K4 A301-503A (1:2,000) (Bethyl), VE-cadherin 2500 (1:2,000), p-JNK1/2 4668 (1:1,000), total JNK1/2 9258 (1:1,000), p-p38 MAPK 4631 (1:1,000), p-IκBα 9246 (1:1,000), IκBα 4814 (1:1,000), p-p65 3039 (1:1,000), p65 8242 (1:1,000), HGK 3485 (1:1,000), p-Erk 4370 (1:1,000) (Cell Signaling Technology), total p38 MAPK sc-535 (1:1,000), total Erk1 sc-93 (1:4,000) (Santa Cruz) or lamin-β1 ab16048 (1:1,000) (Abcam) antibodies. Uncropped western blot images are provided as Supplementary Figs 4–6.