Anti pi4p

The primary antibodies used in this study are as follows: anti-PI(4)P (Echelon Biosciences Inc., Salt Lake City, UT, USA, Z-P004, 10 μg/mL for immunofluorescence (IF), 2.5 μg for immunoprecipitation (IP)), anti-Son (Abcam, Cambridge, UK, ab121759, 1 μg/mL for IF), anti-C23 (Abcam, ab22758, 1 μg/mL for IF), anti-lamin B1 (Abcam, ab16048, 3 μg/mL for IF), anti-PI(4,5)P2 (Echelon Biosciences Inc., Z-A045, 2.5 μg/mL for IF), anti-RPA194 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-28714, 1 μg/mL for IF), anti-mouse IgM isotype control (Abcam, ab91545, 2.5 μg for IP), anti-hnRNP U (Merck, Darmstadt, Germany, 05-1516, 1 μg/mL for Western blot (WB)), anti-NXF1 (Abcam, ab129160, 0.03 μg/mL for WB) and anti-NUMA1 (Abcam, ab109262, 0.1 μg/mL for WB).
The secondary antibodies used in this study are as follows: goat anti-Mouse IgM Alexa Fluor 555 (Invitrogen, Waltham, MA, USA, A21426, 5 μg/mL), goat anti-Mouse IgM Alexa Fluor 568 (Invitrogen, A21043, 5 μg/mL) and goat anti-Rabbit IgG Alexa Fluor 488 (Invitrogen, A11034, 5 μg/mL) for IF; and IRDye^® 800 CW Donkey anti-Rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA, 926-32213, 0.05 μg/mL) and IRDye^® 800 CW Donkey anti-Mouse IgG (LI-COR Biosciences, 926-32212, 0.05 μg/mL) for WB.

Cells were plated on the wells of a 8-well glass-bottom chamber slides coated with collagen. For PIP₃ staining, cells were serum-starved overnight and stimulated with 100 ng/ml for 5 min. Cells were fixed with 3.7% (w/v) paraformaldehyde/0.1% glutaraldehyde for 1 h at 37 °C and permeabilized with 0.15 mg/ml saponin solution. These solutions were prepared in fixation buffer (5 mM KCl, 137 mM NaCl, 4 mM NaHCO₃, 0.4 mM KH₂PO₄, 1.1 mM Na₂HPO₄, 2 mM MgCl₂, 5 mM piperazine-N,N′bis(2-ethanesulfonic acid), pH 7.2, 2 mM EGTA and 5.5 mM glucose. After blocking with 5% (w/v) BSA for 45 min, cells were incubated with primary antibodies (mouse monoclonal anti-PIP₃, anti-PIP₂, and anti-PI(4)P [source: Echelon Biosciences] at a 1:100 dilution) for 1 h at room temperature. After washing 3 to 4 times in PBS, cells were incubated with appropriate secondary antibodies for 45 min and washed 3 to 4 times in PBS before mounting (6 mg/ml propyl gallate prepared in 50% (v/v) glycerol/PBS) and acquisition of fluorescence images. Fluorescence images were taken with either 20× or 60× objective on Olympus wide-field and Zeiss confocal microscopes. All images were background subtracted using ImageJ (https://imagej.net/ij/) software for quantitative fluorescence intensity analyses.

Cells were washed once in PBS before fixation for 20 min in 4% (wt/vol) paraformaldehyde (PFA). The cells were subsequently permeabilized in 0.2% (vol/vol) Triton X-100–PBS before immunostaining with the antibody denoted below. The primary antibodies used were anti-NS5A (sheep, 1:1,000), anti-NS3 (sheep, 1:300; prepared in-house), anti-dsRNA (mouse, 1:200; Scicons), and anti-PI4P (mouse, 1:100; Echelon). Various fluorescently conjugated secondary antibodies were used at 1:1,000 (Life Technology). Note that when conducting immunostaining for dsRNA, all reagents were prepared to be RNase free by DEPC treatment. Lipid droplets were stained using the BODIPY(558/568)-C12 dye at 1:1,000 (Life Technology), which was added at the same time as the fluorescent secondary antibody. Confocal microscopy images were acquired on a Carl Zeiss LSM 700 inverted microscope, and postacquisition analysis was conducted using either Zeiss Zen 2012 or Fiji (v1.49) software (22 (link)).