Ab106582

Anti-A1AR antibody (ab82477), anti-active caspase-3 (ab2302), Chk pAb to albumin (ab106582), Rb pAb to MAPKAP2 (ab131531), Rb pAb to phosphor MAPKAP2 (ab63378), Ms mAb to Hsp27 (ab2790), Rb pAb to phosphor Hsp27 (ab5594), Rb mAb to NeuN (ab177487), anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (ab201015), anti-ERK1 + ERK2 antibody (ab17942), anti-JNK1 (phospho T183) antibody (ab47337), anti-JNK1 antibody (ab110724), anti-GFAP antibody (ab10062), and Ms mAb to NeuN (ab104224) were purchased from abcam. Anti-A2aAR antibody (sc-32261), anti-A2bAR antibody (sc-28996), anti-A3AR antibody (sc-13938), β-actin (sc-47778), and GAPDH (sc-365062) were purchased from Santa Cruz. Rb pAb to P38, phosphor P38 was purchased from Cell Signaling.

Western blot analysis was conducted using an SDS-PAGE electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, the total protein content was extracted from MCF-7, MCF-10A and MDA-MB-231 cell lines using a RIPA buffer (P0013C, Beyotime, Shanghai, China). Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (88,520, Thermo Fisher Scientific, Waltham, MA, USA), which were subsequently blocked 12 h at 4 °C with 5% skimmed milk containing TBST (Tris-buffered salt solution, containing 50 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.1% v/ v Tween-20, pH 7.4) solution. Antibodies against ALB (ab106582, Abcam, San Francisco, CA, USA), ANGPTL7 (orb350273, Biorbyt, Cambridge, UK), BLOC1S5-TXNDC5 (abx230909, Abbexa, Ltd, Cambridge, UK), IL6 (orb10911, Biorbyt, Cambridge, UK.), and NGB (sc-133086, Santa Cruz, Dallas, TX, USA.), and GAPDH (ab8245, Abcam, San Francisco, CA, USA.) were used as primary antibodies. The samples were incubated with horseradish peroxidase–conjugated secondary antibodies at 37 °C for 1 h. The membrane was imaged using an Amersham Imager 600 (GE Healthcare UK Limited, Little Chalfont, UK).

Mice and rats were killed by an overdose of urethane 24 h after dMCAO or craniectomy and then perfused with cold saline to remove circulating albumin from the blood. Isolated brains were coronally sectioned at 2 mm thickness. Motor cortex tissue from the middle two coronal sections (approximately +2 to +6 mm from λ), where infarct occurred or would have occurred, was isolated for Western blotting as described previously (Liao et al., 2019 (link)). In brief, these samples were homogenized in lysis buffer (20 mm Tris, 150 mm NaCl, 1 mm EDTA, and 1% NP-40 adjusted to pH 7.4) containing protease inhibitor (catalog #04693132001, Roche) and centrifuged at 12,000 rpm for 30 min. Proteins (∼30 μg) from the supernatants were heated for 5 min at 100°C, separated by electrophoresis, and transferred to PVDF membranes. After blocking in milk (5% dry milk powder in TBST) for 2 h at room temperature, each membrane was incubated with primary antibodies against α-tubulin (1:5000 in 2% BSA in TBST; catalog #GTX628802, Genetex) and albumin (1:5000 in 2% BSA in TBST; catalog #ab106582, Abcam) overnight at 4°C, washed with TBST, and incubated with goat anti-mouse IgG (1:1000 in 2% BSA in TBST; catalog #GTX213111-01, Genetex) and goat anti-chicken IgY (1:1000 in 2% BSA in TBST; catalog #ab97135, Abcam) secondary antibodies. Western blot images were taken using a ChemiDoc XRS+ System (BIO-RAD).