Mouse anti human gapdh

Proteins were extracted with erythrocyte lysis buffer (ELB; 50 mM Tris, 140 mM NaCl, 0.5% NP-40 and 100 mM NaF (pH 7.6)) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma–Aldrich, USA). Each sample was separated on a 10% SDS-PAGE gel and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk and incubated with primary rabbit anti-human FSCN1 antibody (1:3000; Cell Signaling Technology, USA) or mouse anti-human GAPDH (1:5000; Sigma–Aldrich) antibody overnight at 4°C. The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific). The bands were developed using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Buckinghamshire, UK). The GAPDH protein was used as a loading control.

Primary antibodies used were: mouse anti-human fibronectin (Sigma-Aldrich, F0916), rabbit anti-human fibronectin (Sigma-Aldrich, F3648), rabbit anti-human HSF1 (Santa Cruz Biotechnology, H-311), and mouse anti-human GAPDH (Sigma-Aldrich, G8795). Secondary antibodies used were goat anti-mouse IgG conjugated to HRP (Abcam, ab97023), donkey anti-mouse IgG conjugated to DyLight® 488 (Abcam ab96875) and donkey anti-rabbit IgG conjugated to DyLight® 488 (Abcam ab96891).

Commercial primary antibodies used were: mouse anti-keratin-8 (Sigma-Aldrich, St. Louis, MO, USA), mouse anti-keratin-18 (Sigma-Aldrich), mouse anti-vimentin (Abcam, Cambridge, MA, USA), rabbit anti-SUMO-1 (Abcam), rabbit anti-SUMO-2/3 (Abcam), rabbit anti-Ubc9 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-β-actin (Santa Cruz Biotechnology), and mouse anti-human GAPDH (Sigma-Aldrich). Mouse and rabbit antisera against APH0032 were described previously [33 (link)]. Rabbit antiserum against A. phagocytophilum protein P44 was described previously [34 (link)]. Mouse anti-β-tubulin and Alexa Fluor phalloidin 555 were kind gifts from Jessica Bell (University of San Diego, San Diego, CA, USA) and Lynne Elmore (Virginia Commonwealth University, Richmond, VA, USA), respectively. Secondary antibodies conjugated to Alexa Fluor fluorochromes were obtained from Thermo Fisher Scientific (Waltham, MA, USA).