Rabbit anti gm130

Normal human lung fibroblasts (NHLF) were transduced with lentiviral particles coding for ARF1 WT, ARF1 R99C or empty vector. 48 h later, the samples were washed with PBS and fixed in 4% paraformaldehyde solution (PFA) for 20 min at RT. Next, the cells were permeabilized and unspecific binding was blocked by incubation with blocking solution (3% (w/v) BSA and 0.3% (v/v) Triton X-100 in PBS) for 2 h at RT. The samples were incubated overnight at 4 °C with 1 μg/ml of the primary antibodies rabbit anti-GM130 (Cell Signaling, 12480) and mouse anti-STING (Novus Biologicals, AF6516) dissolved in diluted blocking solution (0.3% (w/v) BSA and 0.03% (v/v) Triton X-100 in PBS). After three washing steps with PBS, the samples were incubated with 1 μg/ml secondary goat anti-mouse antibody conjugated with Atto647N (Sigma-Aldrich, 50185), 1 µg/ml goat anti-rabbit antibody conjugated with Atto594 (Sigma-Aldrich, 77671) and anti-rat antibody conjugated with Alexa Fluor Plus 405 (Thermo Fisher Scientific, A48268, transfection control) dissolved in diluted blocking solution (0.3% (w/v) BSA and 0.03% (v/v) Triton X-100 in PBS) for 1 h at RT. Unbound antibodies were removed in three washing steps with PBS. For imaging, samples were kept in 97% 2,2′-thiodiethanol (TDE, Sigma Aldrich, 166782) solution in PBS, pH 7.5.

The following antibodies and reagents were used: mouse anti-Na,K-ATPase β-subunit (clone M17-P5-F11) (Thermo Scientific, Rockford, IL, USA), rabbit anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Na,K-ATPase α-subunit (Merck Millipore, Darmstadt, Germany), rabbit anti-β-actin (Sigma Aldrich, St. Louis, MO, USA), mouse anti-transferrin receptor (Invitrogen, Rockford, IL, USA), rabbit anti-PDI (Cell Signaling, Danvers, MA, USA), rabbit anti-GM130 (Cell Signaling, Danvers, MA, USA), rabbit anti-calnexin (Abcam, Cambridge, UK), rabbit anti-BiP (Cell Signaling, Danvers, MA, USA), HRP-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling, Danvers, MA, USA), and Alexa Fluor 488 and 594 conjugated anti-rabbit, anti-mouse (Thermo Scientific, Eugene, OR, USA). α-ketoglutaric acid was obtained from Sigma Aldrich, St. Louis, MO, USA.

Cells lysates were prepared in RIPA buffer plus 1:100 protease inhibitor cocktail, phosphatase inhibitor cocktail 2, and phosphatase inhibitor cocktail 3 (all Sigma-Aldrich), and the protein concentration was determined by BCA assay (Thermo Scientific™ Pierce™ Protein Biology, Rockford, IL, USA). Equal volumes of protein (1 or 5 μg) were electrophoresed by SDS-PAGE in a 12 % polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) blots which were blocked in 5 % milk or 5 % bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline + 0.05 % Tween 20 (TBST), and then incubated overnight at 4 °C with either rabbit anti-Akt or anti-serine 473 phosphorylated Akt (p-Akt) (1:1000 in TBST + 5 % BSA; both Cell Signaling Technology), or rabbit anti-Cav-1 (1:5000 in TBST + 2.5 % milk; BD Biosciences, Franklin Lakes, NJ, USA), or rabbit anti-GM130 (1:1000 in TBST + 5 % BSA; Cell Signaling Technology). Secondary antibodies were Amersham ECL donkey anti-rabbit or sheep anti-mouse horseradish peroxidase (HRP)-linked IgG antibody (both GE Healthcare UK Limited, Little Chalfont, UK). HRP activity was detected using Supersignal West Dura, Extended Duration Substrate (Thermo Scientific™ Pierce™ Protein Biology) and the chemiluminescence reaction was visualized using a FOTO/Analyst® Fx CCD imaging system (Fotodyne Inc., Hartland, WI, USA).