Odyssey fc imaging system

For the analysis of protein expression, tissues were collected every 4 h throughout the day and stored at −80°C. For representative Western blotting, three liver samples from three different mice were pooled at each time point. For quantitative data, liver samples were run individually to estimate variability among biological replicates. For lysate preparation, frozen liver pieces were lysed in Cell Signaling lysis buffer with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Beverly, MA) using a sonicator. Protein concentration was determined with the aid of the Bradford protein assay kit according to the manufacturer's protocol, after which lysates were stored at −20°C. Protein (45 μg) was loaded into 3–8% Tris-acetate and 4–12% Bis-Tris gels (Invitrogen). After SDS–PAGE, protein was transferred on the polyvinylidene fluoride membrane at 110 mA. Equal loading of proteins was checked by Ponceau staining. The membranes were probed with the primary antibodies listed in the Supplemental Materials. Probing the same membranes with anti–β-actin or anti–glyceraldehyde-3-phosphate dehydrogenase antibodies was used for normalization of the signal. Images were obtained with Odyssey FC imaging system (LI-COR, Lincoln, NE), and quantification was performed with the Odyssey FC imaging system, version 3.0, software.

Proteins were resolved by SDS-PAGE using 4–20% Mini-Protean TGX gels (Bio-Rad) at 300 V for 20 min and then transferred onto a 0.2-µm nitrocellulose membrane for 60 min at 80 V in a Tris-Glycine transfer buffer with 25% methanol. Blots were blocked in 5% nonfat milk in PBS containing 0.1% Tween-20 for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the blots for 1 h at room temperature or 4°C overnight (4C10G9 mouse anti-mosGILT monoclonal 1:1,000, 0.8 µg/ml; mouse anti-β actin monoclonal, Abcam 8224, 1:1,000; rabbit anti-TEP1 polyclonal 1:200, and rabbit anti-prophenoloxidase 2 [PPO2] polyclonal 1:15,000, two kind gifts from Dr. Elena A. Levashina, Vector Biology Unit, Max Planck Institute for Infection Biology, Berlin, Germany). Infrared fluorescent secondary antibodies, goat anti-mouse IRDye 680LT (926-68020; LI-COR), or goat anti-rabbit IRDye 800CW (926-32211; LI-COR) was diluted in blocking buffer at 1:5,000 and incubated for 1 h at room temperature. Blots were washed in PBS with 0.1% Tween-20 three times for 5 min and then imaged with a LI-COR Odyssey Fc imaging system. Gel densitometry was performed using the in-built quantification tools on the LI-COR Odyssey Fc imaging system and normalized to the PPO2 signal. Data are presented as relative band signal intensity comparing the mosGILT mutants to siblings.

A pooled CRISPR knockout line and comparable wild-type NIH 3T3 cells expressing endogenous IQGAP1 were purchased from Synthego (Redwood City, CA). Cells were grown in DMEM (Gibco, Grand Island, NY) supplemented with 10% FBS (Genesee Scientific, San Diego, CA), 200 mM L-glutamine (Gibco), and 45 U/mL penicillin-streptomycin (Gibco). All cell lines were screened for mycoplasma at regular intervals by screening fixed cells for irregular DAPI stain. Clonal lines were produced via dilution by plating 0.5 cells per 48-well plate (Genesee Scientific) and visual confirmation of single-cell deposition. Wells containing cells were grown in conditioned media, grown to ~ 70% confluency, and screened for protein levels via Western blots with monoclonal mouse anti-IQGAP1 (1:1000; 610612, BD Biosciences, East Rutherford, NJ), polyclonal rabbit α-tubulin (1:2500; ab18251, Abcam Inc, Cambridge, United Kingdom), and appropriate secondary antibodies (1:5000 IR-dye-680 for IQGAP1 and IR-dye-800 for α-tubulin; LI-COR Biotechnology, Lincoln, NE). Blots were imaged with a LI-COR Odyssey Fc imaging system (LI-COR Biotechnology).