Mouse anti gfp

The following antibodies were used for cell division analyses; primary antibodies: rabbit anti-PH3 (Cell Signaling (Danvers, MA) 9701) 1:500; mouse anti-GFP (Cell Signaling 2955) 1:1000. Secondary antibodies: Alexa Fluor 594 donkey anti-rabbit ((A21207) Thermo Fisher Scientific, Waltham, MA) 1:1000; Alexa Fluor 488 donkey anti-mouse (A21202) 1:1000. Guts were dissected in ice cold PBS and immediately fixed in 4% formaldehyde for 15 min, serially dehydrated in MeOH, stored at -20°C, and subsequently stained. Guts were washed in 0.2% Triton-X / PBS, blocked in 5% bovine serum albumin / PBS, incubated in primary antibody overnight at 4°C and in secondary for 2 hr at RT. At least 10 guts per condition were mounted, scored and imaged as described above.

Mouse anti-flag (M2) and mouse anti-β-actin were purchased from Sigma Aldrich. Mouse anti-ubiquitin was purchased from BD. Rabbit anti-flag, mouse anti-GFP, mouse anti-α-tubulin and rabbit anti-caveolin-1 antibodies were purchased from Cell Signaling. Rabbit anti-TfR antibody was a Proteintech Group product. The rabbit polyclonal antibodies for GM130, EEA1 and LAMP1 were all ABclone products. Rabbit anti-TGN46 was Abcam product, Fluor Alexa-488-conjugated goat anti-mouse IgG, Fluor Alexa-555-conjugated goat anti-mouse/rabbit IgG, and Alexa-647-conjugated goat anti-rabbit IgG were all purchased from Life Technologies. IRDye 680LT and 800CW goat anti-rabbit IgG or anti-mouse IgG antibodies were purchased from LI-COR Biosciences. The mouse monoclonal antibodies against K8, ORF45, ORF64, and ORF65, gH were generated in our lab previously.

Approximately 1.2 × 10⁶ HepG2 cells were transfected with 4 μg of human NR4A1 plasmid DNA per electroporation using the Nucleofector Kit V (Amaxa, Gaithersburg, MD, USA), according to the manufacturer’s instructions, and cultured for 30 h. The transfected cells were seeded in 8-well chamber slides. Following a 30-h growth period, the transfected cells were fasted in DMEM with 1% FBS for 16 h. The cells were treated for 2 h with a recombinant fusion protein (20 μg/mL) consisting of human ApoA-IV (r-h-apoA-IV) and green fluorescent protein (GFP), which had been purified as described previously [23 (link)]. The cells were fixed in the chamber slides using 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Non-specific binding was blocked with 5% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated with 1:200 rabbit anti-human NR4A1 (Santa Cruz Biotechnology, Dallas, TX, USA) and 1:200 mouse anti-GFP (Cell Signaling Technology) primary antibodies overnight at 4°C, followed by incubation with 1:200 Alex Flour-594-conjugated goat anti-rabbit (Invitrogen) and 1:200 fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibodies (Invitrogen). The cells were viewed using a Zeiss LSM-510 confocal fluorescence microscope.