9 phenanthrol 9 phe

To detect TRPM4 protein expression, whole-cell lysates were prepared by lysing cells in lysis buffer [50 mM HEPES pH 7.4; 150 mM NaCl; 1.5 mM MgCl 2 , 1 mM EGTA pH 8.0; 10% glycerol; 1% Triton X-100; complete protease inhibitor cocktail (Roche, Mannheim, Germany)] for 1 h at 4°C. Cell lysates were centrifuged for 15 min at 16 000 × g at 4°C, pellets discarded, and protein concentration of the supernatant was evaluated using the Bradford assay. Sixty μg of each protein sample was run on 9% polyacrylamide gels and transferred to a nitrocellulose membrane with the Turbo Blotdry blot system (Biorad, Hercules, CA, USA). After transfer, membranes were blocked with 0.1% BSA in PBS and incubated with rabbit primary anti-TRPM4 antibodies (epitope: VGPEKEQSWIPKIFRKKVC) (generated by Pineda, Berlin, Germany) diluted 1:750 in PBS/Tween using the SNAP i.d. system (Millipore, Billerica, MA, USA), followed by incubation with secondary antibodies (IRDye 800CW 1:1000 in PBS/Tween, LI-COR Biosciences, Lincoln, NE, USA). Membranes were scanned using the LiCor Odyssey Infrared imaging system (LI-COR Biosciences).
Compounds tested 9-phenanthrol (9-Phe) was obtained from Sigma-Aldrich (Buchs SG, Switzerland). Compounds CBA, NBA, and the other anthranilic acid amides (Fig. 7) were synthesised as previously described (Ozhathil et al., 2018) .

Reagents Lipopolysaccharide (LPS) was purchased from Solarbio (Peking, China). Glibenclamide (GLB, an inhibitor of SUR1) and 9-phenanthrol (9-Phe, an inhibitor of TRMP4) were purchased from Sigma-Aldrich (St.Louis, MO, USA). TGFα recombinant proteins were purchased from Abcam (Cambridge, UK). CaMKII-IM-1 (an inhibitor of CaMKII) and KG-501 (an inhibitor of CREB) were purchased from MedChemExpress (NJ, USA). NCGC00067819 (an agonist of CREB) was purchased from Probechem (IL, USA). STAT3 inhibitor VI was purchased from Santa Cruz (CA, USA).