Hexokinase 1

Cell lysates from H460, A549, H1975, and HCC827 cells were prepared 24 h after treatment with DMSO, Milciclib (1 or 10 μM), THZ1 (10 μM), or LDC4297 (10 μM); 16 h after treatment with 1× PBS or Deferoxamine (100 μM); or 72 h after transfection with shRNA or an overexpression plasmid. Uncropped immunoblots are presented in Supplementary Fig. 29.
The following antibodies were used: HIF-1α (Novus Biologicals; NB100-105; 1:1000 dilution), GLUT1 (Millipore Sigma; 07-1401; 1:500 dilution), GLUT3 (Abcam; ab15311; 1:1000 dilution), Hexokinase 1 (Cell Signaling; C35C4; 1:1000 dilution), Hexokinase 2 (Cell Signaling; C64G5; 1:1000 dilution), CDK2 (Cell Signaling; 78B2; 1:1000 dilution), CDK4 (Cell Signaling; D9G3E; 1:1000 dilution), CDK7 (Cell Signaling; MO1; 1:1000 dilution), TRKA (Cell Signaling; 12G8; 1:1000 dilution), PIK3CA (Cell Signaling; C73F8; 1:1000 dilution), PTEN (Cell Signaling; 9559; 1:1000 dilution), Phospho-T170 CDK7 (Abcam; ab155976; 1:1000 dilution), Rpb1 NTD (Cell Signaling; D8L4Y; 1:1000 dilution), Phospho-S5 Rpb1 (Cell Signaling; D9N5I; 1:1000 dilution), PKCι (BD Biosciences; Clone 23; 1:1000 dilution), TXNIP (Cell Signaling; D5F3E; 1:1000 dilution), and Beta Actin (Imgenex; IMG-5142; 1:1000 dilution). Immunoblots were analyzed using an Odyssey Imaging System (LI-COR).

All chemicals were purchased from Sigma-Aldrich, unless indicated otherwise. Rabbit polyclonal antibody against HIF-1α was obtained from (Novus Biologicals, Littleton, CO, USA, #NB100–449), while phosphorylated mTOR S2448, β-actin, α-Tubulin, as well as Glycolysis Antibody Sampler Kit to detect Pfkp, Pkm2, Pkm 1/2, Hexokinase 1, Hexokinase 2 and Pdh were all obtained from (Cell Signaling Technology, Beverly, MA, USA, #2971, #4970, #2148, #8337 respectively). Secondary antibodies consisted of horseradish peroxidase conjugated goat anti-rabbit IgG (Agilent’s Dako, Glostrup, Denmark). Torin 1, LY294002 and Forskolin were purchased from (ApexBio, Houston, TX, USA). ON-TARGETplus Rat Hif1a siRNA – SMARTpool (siHif1a) and Non-targeting Pool siRNA (siControl) were purchased from (Dharmacon, Lafayette, CO, USA - L-091718-02 and D-001810-10-05, respectively).

Polyclonal LDHA, LDHB, and aldolase C antibodies were purchased from Proteintech (Rosemount, IL). Rabbit polyclonal red/green cone opsin (M-opsin), S-opsin, cone arrestin, actin, and rabbit and mouse secondary antibodies were obtained from Millipore (Billerica, MA). Monoclonal 1D4 rhodopsin antibody was a kind gift from Dr. James F. McGinnis (University of Oklahoma Health Sciences Center). DAPI used for nuclear staining was procured from Invitrogen-Molecular Probes (Carlsbad, CA). Polyclonal pPKM2 (Y105), PKM2, PKM1, PDH, hexokinase 1, and hexokinase 2 antibodies were obtained from Cell Signaling (Danvers, MA). The monoclonal anti-arrestin antibody was a kind gift from Dr. Paul Hargrave (University of Florida, Gainesville). Polyclonal glial fibrillary acidic protein (GFAP) was purchased from Dako (Carpinteria, CA). Monoclonal GS antibody was purchased from Abcam (Cambridge, MA). The monoclonal Pde6β antibody was purchased from Santa Cruz Biotechnology (Dallas, TX).

Whole cell lysates were extracted with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland) and subjected to Western blot analysis as previously described 10. Proteins were separated on 4–15% gradient polyacrylamide–sodium dodecyl sulfate gels (Bio‐rad Laboratories, Inc., Hercules, CA), transferred to Supported Nitrocellulose membranes (Bio‐rad) using a Bio‐Rad Mini‐PROTEAN Tetracell system, followed by incubation for 1 h in a bovine serum albumin‐Tween‐20‐based blocking solution. Primary antibodies were Hexokinase I (1:1000), Hexokinase II (1:1000), phosphofructokinase (PFK) (1:1000), pyruvate kinase M1/2 isoform (PKM1/2) (1:1000), pyruvate kinase M2 isoform (PKM2) (1:1000), pyruvate dehydrogenase (PDH) (1:1000), lactate dehydrogenase A (LDHA) (1:1000), GAPDH (1:1000), and actin (1:1000), all from Cell Signaling Technology, Inc.(Danvers, MA). Blots were incubated with primary antibody overnight and then incubated for 1 h with horseradish phosphatase–conjugated anti‐rabbit or anti‐mouse secondary antibodies (1:10,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Blots were developed using the Enhanced Chemiluminescence Kit (Pierce, Thermo Fisher Scientific, Waltham, MA) followed by autoradiography.