Thermo scientific subcellular protein fractionation kit

In experiments of Figs. 2A, B, 3F, and 6J, the cells were subjected to Thermo Scientific Subcellular Protein Fractionation Kit (cat. No. 78840, Thermo) for separating cytosolic, total membrane, and nuclear fractions, following the manufacturer’s instructions. In experiments of Fig. 2E, the cells were subjected to BioVision Plasma Membrane Protein Extraction Kit (cat. No. K268-50/ab65400, BioVision) for separating plasma membrane and cellular organelles (non-plasma membrane from the bottom phase of step B4 of the manufacturer’s instructions), following the manufacturer’s instructions. In experiments of Fig. 6G, the cells were subjected to Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (cat. No. SM-005, Inventbiotech) for isolating plasma membranes and subjected to Minute™ Endosome Isolation and Cell Fractionation Kit (cat. No. ED-028, Inventbiotech) for isolating endosomes, following the manufacturer’s instructions.

To detect various viral and cellular protein expressions, ACH2 cells were treated with PKC412 for 12–48 h, and cells were lysed with RIPA buffer and run on a 12 % SDS-PAGE gel, followed by immunoblotting with various antibodies, including anti-HIV-1 gp120, anti-HIV-1 p24, anti-tubulin, anti-acetyl-histone H3, anti-histone H3, anti-NF-κB p65, or anti-P-NF-κB p65 (Ser³¹¹). The protein bands were visualized using an enhanced chemiluminescence kit (Perkin Elmer Life Science, Boston, USA).
For subcellular protein fractionation and detection, a Thermo Scientific Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, USA) was used to prepare cytoplasmic, nuclear fractions from ACH2 cells, according to manufacturer’s recommended procedures. Then, each subcellular fraction was subjected to Western Blotting to detect NF-κB p65, Histone-4, β-tubulin by using specific antibodies.
For the indirect immunofluorescence assay, ACH2 cells were treated with PKC412 and then plated on a slide, fixed and permeabilized with methanol/acetone (1:1 ratio) for 30 min at room temperature. The cells were incubated with the mouse anti-p24 antibody (1:100) for 2 h at 37 °C, followed by incubation with an anti-mouse IgG-488 antibody (1:500) for 1 h. The slide was mounted with Mowiol 4–88 and the cells were visualized under an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany).

Two hundred and ninety-three T cells were transfected with AcGFP-IN wt or 3VI in 6-well plate for 48 h. Cells were harvested and proteins were sequentially extracted, yielding cytoplasmic, nuclear and chromatin-bound fractions using a Thermo Scientific Subcellular Protein Fractionation Kit (Thermo Scientific, USA). Each fraction of proteins was subject to WB analysis using the anti-GFP antibody.