Image quant las 4000 platform

SDS-PAGE and immunoblot
analysis were performed as described previously.^{23 (link),31 (link),52 (link)} Protein concentrations were quantified by
the Bradford method.^{53 (link)} Anti-RbcL (1:10,000
dilution, Agrisera, Sweden), anti-CsoS1 from H. neapolitanus (1:5000 dilution, Agrisera, Sweden), and anti-HisTag (Invitrogen,
USA) antibodies and horseradish peroxidase-conjugated goat antirabbit
immunoglobulin G secondary antibody were used for immunoblot analysis
and imaged on an Image Quant LAS 4000 platform (GE Healthcare Life
Sciences, USA).

SDS-PAGE gels were cast using a pre-made solution of acrylamide:bisacrylamide :: 29:1. Cell lysates were loaded on gels, in most cases at 30 µg total protein per lane. The resolved proteins on the gels were transferred to PVDF membranes (FluoroTrans^® W, 0.2 µm pore size, Pall Corporation, New York, USA) by semi-dry transfer at constant current. Recipes for separating and stacking gels, gel running buffer and transfer buffer were standard recipes from (Green and Sambrook, 2014 ). Membranes were blocked in 5% fat-free milk prepared in TBST (Tris-buffered saline with Tween 20; 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20). Primary antibodies were added according to the concentration provided by the manufacturer (usually a dilution of 1:1000–1:5000) and secondary HRP-conjugated antibodies were used at a dilution of 1:10,000. The blots were developed using Immobilon^® chemiluminescent HRP substrate (EMD Millipore, Merck), on an ImageQuant LAS 4000 platform (GE Healthcare). Quantitation of western blots was done using the software ImageJ, and graphs were plotted using GraphPad Prism, version 6 (GraphPad Software, California, USA). Blots were stripped using a buffer comprising 62.5 mM Tris-HCl pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol, and re-probed for loading controls and other proteins.