Goat anti rabbit ir800

Protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and wet transferred onto a nitrocellulose membrane. Membranes were probed with the following primary antibodies at a 1:1000 dilution: targeting S555 p-ULK1 (CST #5869), S757 p-ULK1 (CST #6888), ULK1 (Sigma-Aldrich #A7481), Vdac (CST #4866), OXPHOS cocktail (Abcam #110413), and Gapdh (CST #2118). The secondary antibodies were goat anti-mouse IR680 and goat anti-rabbit IR800 (LICOR). Membranes were scanned using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). Proteins were analyzed in comparison to a common protein standard loaded on the gel, prior to calculating phospho:total, signal:Ponceau, or protein:Gapdh ratio.

BMDMs were obtained as above. Peritoneal cells were obtained by injecting 5 ml PBS with 5% FBS into the peritoneum, and then massaged to dislodge cells. Fluid was then collected with needle syringe and confirmed for the absence of blood contamination. Total cell lysates from 1e+6 cells were obtained by adding 0.02% NP-40 supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific) and 1X SDS-PAGE buffer (0.0625M Tris-Cl pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, 5% 2-Mercaptoethanol, 0.01% bromophenol blue) and loaded into 10% SDS-polyacrylamide gels. Gels were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore) and probed for CK2α (Biomatik) and β-Actin (8H10D10; Cell Signaling), followed by secondary goat anti-rabbit IR 800 (926-32211; LI-COR) and goat anti-mouse IR 680 (926-68070; LI-COR). Blots were imaged on an Odyssey CLX (LI-COR). Expression of CK2α was normalized to β-Actin using Image Studio Lite ver5.2 (LI-COR) software.

Quantitative RT-PCR (qPCR) and immunoblots were performed as before [14 (link)]. We used the following taqman probes for qPCR analysis: HUMAN—NRF2 (Hs00975961_g1, Life Technologies, Carlsbad, CA, USA), FN3K (Life Technologies, Hs_00223368_m1), GUSB (Life Technologies, 4333767 F), and ACTB (Life Technologies, 4332645); MOUSE—Nqo1 (Life Technologies, Mm01253561_m1), Nrf2 (Life Technologies, Mm00477784_m1), Fn3k (Life Technologies, Mm00445584_m1), cMyc (Life Technologies, Mm00487804_m1) and Actb (Life Technologies, Mm00607939_s1). For immunoblotting, we used the following antibodies from Cell Signaling (Danvers, MA, USA): NQO1 (CST, 62262), MYC (CST, 5605), BCL2 (CST, 3498), KEAP1, (CST, 8047), CUL3 (CST, 2759), GAPDH (CST, 5174), and β-actin (Sigma-Aldrich A5441 St. Louis, MO, USA). Proteins were visualized using LI-COR Lincoln, NE, USA detection system after incubation with following secondary antibodies: Goat anti-rabbit-IR800 (LI-COR, 926–32211) and Goat anti-mouse-IR680. We used the following reporter assays as per manufacturer’s instructions: Luciferase-Glo (Promega) and Beta-glo (Promega). T7 endonuclease assays were conducted to confirm gene editing (NEB, Ipswich, MA, USA).

Tissues homogenates were prepared as previously described [30 (link)]. Thirty μg of total protein was subject to SDS-PAGE and transferred onto PVDF membrane. Membranes were probed with the following primary antibodies: mouse anti-CypD (AbCam, ab110324), rabbit anti-ANT (AbCam, ab180715), rabbit anti-VDAC (CST, #4866), mouse anti-ATP Synthase (Abcam, ab5432), mouse anti-phospho-Akt S473 (CST, #4051), rabbit anti-Akt (CST, #4691), mouse α-Tubulin (Abcam, ab11304). Secondary antibodies were goat anti-mouse IR680 (LICOR) and goat anti-rabbit IR800 (LICOR). Membranes were scanned using the Odyssey infrared imaging system (LICOR).

Larvae were incubated with 10 mM ANL for 24 hr. After incubation, the media was replaced with fresh E3. Larvae were sacrificed on ice-cold water (30 min). Heads were dissected using a scalpel and immediately snap frozen in tubes (1.5 ml, Eppendorf) that were pre-cooled using dry ice and stored at −80C until lysis. Tissue was homogenized and lysed using a pestle in lysis buffer (1% in Triton X100, 0.4% (w/v) SDS in PBS pH 7.8, 1:1000 EDTA free protease inhibitors (Calbiochem) and benzonase (Sigma, 1:1,000), and denatured at 75°C, 13,000 rpm for 10 min. Lysates were then cleared by centrifugation. BONCAT was performed as previously described (Dieterich et al., 2007 (link)). In brief, 60 μg proteins were dissolved in 120 μL PBS pH 7.8 supplemented with 0.01% SDS, 0.1% Triton, 300 μM Triazol (Sigma, 678937), 50 μM biotin-alkyne tag (Thermo, B10185) and 83 μg/mL CuBr (prepared by dilution of fresh 10 mg/mL solution in DMSO) at 4°C overnight in the dark. Biotinylated proteins were then separated by gel electrophoresis and immunoblotted with 1:1000 chicken anti-GFP (Aves), 1:1000 rabbit anti-biotin (Cell signaling) and Donkey anti-chicken IR680, goat anti-rabbit IR800 (IB, 1:10,000, Licor) antibodies.