Anti gck

Protein samples were extracted from cells using RIPA buffer (50 mmol/L Tris‐HCl at pH 7.4, 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, 1 mmol/L EDTA) containing protease and phosphatase inhibitor cocktails (Calbiochem). They were then subjected to Tris‐Glycine PAGE (Invitrogen), immunoblotted with rabbit anti‐CB1R (1:500; Frontier Science), anti‐insulin (1:1000; Santa Cruz), anti‐GCK (1:500; Abcam), anti‐GLUT2 (1:1000; Santa Cruz) or mouse anti‐β‐actin (1:10000; Abcam), and visualized by ECL (GE Health). Whole pancreata were homogenized by a motor‐driven pestle in ice‐cold RIPA buffer containing protease and phosphatase inhibitor cocktails. Homogenates were solubilized by end‐over‐end mixing at 4°C for 60 minutes and subjected to centrifugation. Total protein was determined using Bradford assay (Bio‐Rad).

A Cell Counting Kit-8 (CCK-8, Cat# CK04) was purchased from Dojindo (Kumamoto, Japan). Pooled antisense oligonucleotides (i.e., siRNAs) against human OVOL2, P65, and GLUT1 were purchased from GenePharma (Shanghai, China). Antibodies against OVOL2 (Cat# ab101580) and P65 (Cat# ab16502) were purchased from Abcam (Cambridge, MA, USA). The following antibodies were used: Glycolysis Antibody Sampler Kits (Cell Signaling Technology; Cat# 12866 and Cat# 8337), anti-GLUT1, anti-GLUT2, anti-GLUT3, anti-GLUT4, anti-PGK1, anti-GCK, anti-PDP2, anti-DLD, anti-PCK1, anti-SDHA, anti-G6PD (all from Abcam; Cat# ab115730, ab234440, ab191071, ab188317, ab38007, ab184169, ab99170, ab133551, ab28455, ab14715, and ab210702, respectively), anti-GAPDH anti-Lamin B, anti-actin and anti-Calnexin (all from Cell Signaling Technology; Cat# 8884, 13435, 3700 and 2433, respectively).

Rabbit polyclonal antibodies against FASN (#3180), PGC-1α(#2178), SCD1(#2438), ACLY(#4332), p-p38 MAPK(#4511), ACC(#4190), P-AMPKα1 (#2537), p-mTOR-S244–8(#5536), mTOR(#2983), and α-tubulin(#2144) were from Cell Signaling Technology. Anti-PDK4(#DF7169) antibodies were from Affinity Biosciences. Anti-GCK(#ab137714), anti-CD36 (#ab133625), and Anti-PDK4(#ab89295) antibodies were from Abcam. Anti-NR1H4(#PP-A9033A-00) was from R&D Systems. Anti-SREBP-1c (#Sc-13551) was purchased from Santa Cruz. Sodium dichloroacetate (#347795) was purchased from Sigma-Aldrich. Sodium oleate/sodium palmitate (#SYSJKJ006) was purchased from Xi'an Kun Chuang Technology Company. All other chemicals were from Sigma-Aldrich.