Hrp labeled secondary antibody

A Western blot was performed to assess the concentration of pro-angiogenic proteins in the PLT-releasate of 4 HDs and 6 GBMs. Specifically, the protein amount was measured by Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific). An equal amount of protein (50 µg) was separated in Bolt 10% Bis-Tris Plus Gel (Thermo Fisher Scientific) in a Mini Gel Tank (Thermo Fisher Scientific) and transferred onto nitrocellulose iBlot 2 Transfer Stacks using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). After transfer, the membrane was blocked in Tris-buffered saline/Tween 20 + 5% milk solution and incubated separately with anti-GAPDH (SantaCruz Biotechnology), anti-VEGF (Abcam), anti-VEGFR2 (Abcam), anti-VWF (Thermo Fisher Scientific), or anti-S1P (Sonepcizumab, Creative Biolabs) overnight at 4 °C. After incubation with HRP-labeled secondary antibody (Invitrogen, Carlsbad, California, USA), the protein bands were scanned using SuperSignal West Femto PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and detected by iBright5000 ChemiDoc XRSþ (Thermo Fisher Scientific). Densitometric analyses were performed using ImageJ [21 (link)].

BSA and citBSA protein samples (2 µg) and PageRuler Plus Prestained Protein Ladder (molecular weight marker) were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were then blocked with 5% skim milk at room temperature for 2 h, washed once with deionized water, and reacted with anti-citrulline antibody, include 22F1,30G2, and MABN328 (Millipore) (diluted in skim milk solution to 1 µg/mL) at room temperature for 1 h. After washing with PBST 5 times, HRP-labeled secondary antibody (Invitrogen, 31440, diluted in skim milk solution to 1 µg/mL) was added for incubation at room temperature for 1 h. After washing again with PBST 5 times, WesternBright Sirius chemiluminescent detection kits were used to reveal the immunoreactive bands and images developed using the ChemiDoc™ MP Imaging System.

To investigate the effects of SIRPα variants on the signaling pathways after ICH, Western blotting was performed as described previously [15 (link)]. Briefly, primary cultured microglia grown in 6-well plates were harvested 24 h after administration with or without erythrocytes and then homogenized in cold RIPA buffer (Cell Signaling Technology) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor cocktail (1:50, Sigma-Aldrich). Simultaneously, the homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected for protein detection. Twenty-five micrograms of proteins were loaded into each lane and subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 4 ~ 15% Ready Gel (Bio-Rad Laboratories Inc.) at 100 V for 120 ~ 180 min. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) at 250 mA for 2 ~ 4 h. The PVDF membranes were incubated overnight with primary antibodies at 4 °C, followed by HRP-labeled secondary antibody (Invitrogen) for 1 h at room temperature. All the primary antibodies used are listed in Table S2. Membranes were scanned using the Typhoon Trio System (GE Healthcare). The optical densities of all protein bands were analyzed by IMAGEQUANT 5.2 software (GE Healthcare).