Gapdh ab

Suspended cells were collected, spun down, washed with cold PBS and lysed in 100 µl Novex Tris-Glycine SDS sample buffer (Thermo Fisher Scientific; LC2676). Adhered cells were washed with cold PBS, lysed in another 100 µl sample buffer, collected and combined with lysates from suspended cells. After sonication, samples were centrifuged at 20,000 g for 10 min at 4°C. Equal amounts of protein samples, measured with Nanodrop (Thermo Fisher Scientific), were denatured using DTT and heating at 95°C for 10 min followed by size separation on a 10% Mini-PROTEAN gel (Bio-Rad; 4561033). Proteins were transferred to PVDF membranes (Bio-Rad; 1074156) using the Trans-Blot Turbo system (Bio-Rad) after which membranes were blocked in TBST-5% milk. Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C. Thereafter, membranes were shortly washed and incubated with HRP-labelled secondary Abs (1:5000; Dako) for 1 h at room temperature. After thorough washing with TBST, protein bands were visualised using Western lightning ECL (PerkinElmer; NEL1030001EA) and the ChemiDoc Touch Imaging System (Bio-Rad).