Calyculin a

pEGFP-C1 F-tractin-EGFP was a gift from Dyche Mullins (Addgene plasmid # 58473) (Belin et al., 2014 (link)). The tdTomato-F-tractin plasmid was a gift from Dr. John A. Hammer and the MLC-EGFP plasmid was a gift from Dr. Robert Fischer, National Heart, Lung, and Blood Institute. Latrunculin A was purchased from Sigma Aldrich Calyculin A was purchased from Cell Signaling Technology, Y-27632 was purchased from Selleck Chemicals, and dimethyl sulfoxide (DMSO) was purchased from Thermo Fisher Scientific.

(RS)-3,5-Dihydroxyphenylglycine (DHPG), NNC 55–0396 dihydrochloride (NNC), nimodipine, 2-aminoethoxydiphenylborane (2-APB) and cycloheximide were obtained from Tocris. Rapamycin and Calyculin-A were from Cell Signaling. Calpain inhibitor III was from Calbiochem. Okadaic acid was from Santa Cruz. All the other reagents were from Sigma. Water insoluble compounds were dissolved in DMSO and diluted to reach a final concentration less than 0.1%.

The quantity of IP-10-expressing hepatocytes was assessed by Western blot analysis. To this end, the isolated hepatocytes were counted and equal cell numbers were re-suspended in lysis buffer containing protease inhibitor cocktail (3 µl/ml, Sigma) and calyculin A (100 nM, Cell Signaling) followed by 10 min incubation on ice with repeated vortexing. Protein sample buffer (4x concentrated) was subsequently added (40% glycerol; 240 mM Tris; HCl pH 6.8; 8% SDS; 0.04% bromophenol blue; 7% β-mercaptoethanol) and the samples were denatured at 99 °C for 5 min. Samples were stored at −20 °C and denatured again at 99 °C for 5 min upon anew addition of 7% β-mercaptoethanol. The samples were separated using a 15% SDS-polyacrylamide gel followed by electroblotting onto a nitrocellulose membrane. Membranes were incubated first with the primary antibodies against IP-10 or α-tubulin (goat anti mouse IP-10, R&D Systems; rabbit anti mouse α-tubulin, Abcam) at 4 °C overnight and second with the respective horseradish-peroxidase-conjugated antibodies. The enzymatic reaction was detected using the ECL System (Santa Cruz Biotechnology) and the images were acquired by the ChemiDoc-it System (Bio-Rad). The relative concentration of IP-10 (ratio of volume under the integral of IP-10 and α-tubulin) was assessed using Image Lab Software (5.2.1 Bio-Rad).