Analyzing Integral Membrane Proteins

Protein extracts from cell lines and tissues were prepared and analysed as previously reported.^{32 (link)} Antibodies to calreticulin (ab2907), TAPBP (ab140982), ERP57 (ab13506) and MHC class I (ab70328) were from Abcam (Cambridge, MA, USA); PPARγ (sc-7273), TRAP1 (sc-9134), GRP78 (sc-13968), anti-mouse (sc-2031) and anti-rabbit (sc-2004) were from Santa Cruz Biotechnology (Dallas, TX, USA); ANXA1 (71–3400) from Thermo Fisher, β-Actin (F-3022) from Sigma-Aldrich (Milan, Italy); E-cadherin (BD 610405) from BD transduction (BD Biosciences, San Jose, CA, USA). To analyse surface proteins, we used an extraction method based on a published procedure. Positivity for E-cadherin, a plasma membrane protein, and negativity for β-Actin, a cytosolic protein, proved that the identified proteins were truly integral membrane components. Comassie blue staining was also used for assessing equivalent protein load. Proteins were then analysed by western blotting as previously reported.^{32 (link), 33 (link)}