Biotin labeled aal

Ninety-six–well microtiter plates were coated with 2 mg/mL anti–human IgG in coating buffer overnight. Plates were then blocked with deglycosylated blocking buffer (deBSA; BSA treated with sodium periodate to oxidize any sugars present). After blocking, serum IgG was diluted at 1:1000 in TBS-Ca-Mg and 0.05% Tween-20 (Thermo Fisher Scientific) and incubated at 37°C for 2 hours. These serum dilutions resulted in a saturating binding of IgG to the ELISA plate. After every incubation step, the plates were washed 3 times with 200 mL TBS-Ca-Mg-Tween. To detect exposed glycosyl residues on IgG, biotin-labeled AAL (50 ng/mL, Vector Laboratories) was added at room temperature for 1 hour. After washing, the plates were incubated with HRP-streptavidin (The Jackson Laboratory) at the recommended concentration for 1 hour at room temperature. Detection was performed for both types of ELISA with the addition of substrate solution. Optical density values were obtained by ELISA-plate reader employing a 450 nm/620 nm filter/reference pair. This method was adapted from prior studies (113 (link)).

Ox-LDL was purchased from Yiyuanbiotech (YB-002, Guangzhou, China). The anti-CDC42 antibody, anti-Rac1/2/3 antibody, anti-PAK antibody, anti-pCDC42 antibody, anti-pPKA antibody, and Phalloidin-iFluor 488 were purchased from Abcam (Oxford, UK). The anti-Unc5b antibody was purchased from CST (Danvers, USA). The PAK inhibitor (FRAX486) and CDC42 inhibitor (ML141) were purchased from MCE (MCE, USA). Biotin-labeled LTL, biotin-labeled LCA, biotin-labeled AAL, biotin-labeled UEA1, biotin-labeled SNA, biotin-labeled MAL1, biotin-labeled VVL, and biotin-labeled PHA-L were purchased from Vector Laboratories (Peterborough, UK). ER-Tracker Blue‒White DPX was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Oil Red O solution was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). FBS was purchased from BI (Israel).