Anti rabbit hrp

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin/streptomycin were purchased from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Anti-phospho-Akt, anti-total Akt, anti-phospho-Foxo and anti-Foxo antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-mouse HRP and anti-rabbit HRP were purchased from Thermo Scientific. PVDF membranes and SuperSignal® West Pico and West Femto Chemiluminescent Substrate for western blotting analysis were purchased from Thermo Scientific, and X-ray films from AGFA (Mortsel, Belgium). SYBR Green Master Mix for the real-time PCR analysis was purchased from Applied Biosystems (Foster City, CA, USA).

Tissue homogenates of yolk sac (E9.5) and adult liver (male β-kl+/− mice, 12 wk of age) were prepared in buffer (20 mM 4-[2- hydroxyethyl]-1-piperazineethanesulfonic acid, 150 mM NaCl, and 0.5% Nonidet P-40 [pH 7.4]) containing protease inhibitor cocktail (cOmplete, Mini, EDTA-free; Roche), and phosphatase inhibitor cocktail (Nacalai tesque). The supernatant was electrophoresed on 4–20% gradient SDS-polyacrylamide gel and transferred to PVDF membranes. After blocking with 5% skim milk in TBS-T for 1 h at room temperature, the membrane was incubated overnight with goat anti-mouse KLB polyclonal antibody (AF2619, 1:1,000; R&D Systems) at 4°C. Then, the membrane was incubated with anti-goat HRP (705-035-147, 1:100,000; Jackson) for 45 min at room temperature. The signals were developed by using the ECL Prime Western Blotting Detection Reagents (Cytiva) and detected with a FUSION SYSTEM (Vilber-Lourmat). After stripping, the membrane was reprobed with rabbit anti-β-actin monoclonal antibody (4970, 1:1,000; Cell Signaling) and anti-rabbit HRP (A16110, 1:200,000; Thermo Fisher Scientific).

About 100 dechorionated sfGFP-dBrd4 embryos between 0.5 and 1.5 h old were transferred to 50 μl of RIPA buffer supplemented with protease inhibitors (Pierce, #A32955). Embryos were homogenized on ice by a plastic pestle, and then 50 ul of 2X SDS-PAGE sample buffer was added. After boiling for 5 min, 10 ul samples were separated on 7.5% SDS-PAGE at room temperature and then transferred to PVDF membrane in a cold room. The membrane was blocked with TBST (0.1% tween 20) and 5% milk, blotted with 1:5000 rabbit anti-GFP (Abcam, #ab290) in a cold room overnight, followed by 1:20000 anti-rabbit HRP (Thermo Fisher, #A16096) at room temperature for 1 h. The membrane was incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, #34577) and exposed to film (Thermo Fisher, #34091).

Total protein extracts were prepared using a lysis buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.74 M beta-mercaptoethanol), sonicated on ice using a Sonifier 250D (Branson Ultrasonics), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore). Antibodies used were as follows: MYC #ab32 (Abcam, Cambridge, USA) [56 (link)], #sc764 (Santa Cruz Biotechnology, USA) [57 (link)]; DNMT3B #ab122932 (Abcam, Cambridge, USA) [58 (link)],#nb100-56514 (Novus Biologicals, USA) [59 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, USA) [22 (link)]; β-ACTIN #A5441 (Sigma-Aldrich, USA); GAPDH #sc25778 (Santa Cruz Biotechnology, USA) [60 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, USA) [61 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, USA) [62 (link)]. Protein quantitation was performed using ImageJ software (https://imagej.nih.gov/ij/) [63 (link)].

Protein lysates were prepared with ice-cold RIPA buffer (Boston Bio Products) supplemented with protease and phosphatase inhibitors (Roche). Protein lysates were separated using SDS-polyacrylamide gel electrophoresis (pre-cast gels, Thermo Fisher) and transferred to a nitrocellulose membrane. After blocking the membrane with 5% milk in PBST (PBS with 0.1% Tween-20, Sigma), the membranes were incubated overnight at 4 °C with anti-GLUT1(Novus, Fgi.72) and anti-Beta Actin (Bethyl, Cat #A300-485A) antibodies, diluted in PBST with 5% BSA to 1:1000. The blots were washed 3 times and then incubated with secondary antibodies (anti-Rabbit HRP, Thermo Fisher), diluted in PBST with 5% milk. Finally, the membranes were incubated with ECL substrate (Pierce) for 1 min and exposed for signal detection with the iBright Imager (Thermo Fisher).

Total lysate equivalent to 1 × 10⁶ cells was loaded on 10% SDS-page electrophoresis and run in 80 V–120 V. Protein transfer to membrane was performed at 330 mA for 1 h. For KINDLIN3 detection, rabbit anti-KINDLIN3 was kindly provided by the Moser lab (9 (link)). SKAP2 detection was achieved by using the SKAP2 polyclonal antibody (PA5-54963, Invitrogen), and TALIN1 was detected using mouse TALIN1 antibody (Abcam). CD18 was identified using CD18 antibody kindly provided by the Fagerholm lab (50 (link)). HA-tag was detected by using the rabbit anti-HA antibody (clone 12CA5, Roche). GAPDH or α-tubulin was used as loading control (EMD Millipore). Incubation of the antibody mix was performed using 2.5% ELK (Campina) overnight at 4°C end over end. Secondary antibodies used were the following: anti-rabbit HRP 1:3,000 (Thermo Fisher Scientific), anti-mouse IgG IRDye 700 1:3,000, anti-mouse IRDye IgG 800 1:3,000 (LICOR), and donkey anti-mouse IgG IRDye 800 antibody (LICOR). Protein expression was visualized after incubation with the SuperSignal West Dura substrate (Thermo Fisher Scientific) in a medical film processor (SRX-101A) or using the Odyssey (LI-COR Biosciences, Lincoln).