Anti anxa2

Cells were washed with PBS, and total proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris–HCl, pH 7.5; 150 mM sodium chloride; 0.5% sodium deoxycholate; 1% Nonidet P-40; 0.1% sodium dodecyl sulfate) supplemented with phosphatase and protease inhibitor cocktail (Millipore Corporation, Burlington, MA, USA). Cell lysates were cleared by centrifugation and subjected to immunoblot analysis. Antibodies included anti-AnxA2 (BD Pharmingen #610069), anti-GAPDH (Santa Cruz Biotechnology #sc32233), and ant-β-actin (Santa Cruz Biotechnology #sc47778). The intensity of protein bands was quantified by densitometry using ImageJ software.

Cells were lysed using RIPA buffer (Thermo Fisher) with Halt protease inhibitor (Thermo Fisher). Protein was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher). Samples were run on a 10% Bis-Tris agarose gel with MES (Thermo Fisher). Gels were transferred using the iBlot2 mini system (Thermo Fisher) and were blocked in PBS Starting Block Blocking Buffer (Thermo Fisher) prior to primary antibody staining overnight at 4 °C with anti-anxA2 (BD Biosciences, San Jose, CA, USA), anti-S100A10 (BD Biosciences), anti-langerin (Cell Signaling Technology, Danvers, MA, USA), anti-beta-actin (Cell Signaling Technology), or GAPDH (Cell Signaling Technology). Secondary antibodies, anti-mouse IRDye 800CW (LI-COR, Lincoln, NE, USA), and anti-rabbit (H+L) Alexa Fluor 680 (Thermo Fisher), were added for 1 h at room temperature. Membranes were imaged using the Odyssey imaging system (LI-COR) and protein images were analyzed and quantified using Image Studio Lite software (version 5.2.3, LI-COR).