Ab43014

J-Lat cells were cultured in the presence (P/I) or absence (CM) of mitogens for 6 h. Whole protein extracts were then digested with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) supplemented with protease inhibitors (Sigma-Aldrich). Extracts before immunoprecipitation were used as controls to ensure that all samples contained the same starting material (input). Extracts were subjected to immunoprecipitation with a rabbit polyclonal anti-Tat antibody (ab43014, Abcam) (0.1 μg/mL) using magnetic protein G beads (Dynabeads, Invitrogen). Protein–protein interactions between the transcription factors NFAT2 and Tip60 and HIV-1 Tat were examined using Western immunoblotting with a mouse monoclonal antibody to NFAT2 (sc-7294, Santa Cruz) and a mouse monoclonal antibody to Tip60 (sc-166323, Santa Cruz). An extract of J-Lat cells expressing NFAT and Tip60 without antibody (-Ab) was used as a negative control. A goat anti-rabbit IgG HRP-conjugated antibody (sc-2004, Santa Cruz) and a goat anti-mouse IgG HRP-conjugated antibody (sc-2005, Santa Cruz) were used as secondary antibodies.

The following primary antibodies were used for immunoblot analysis: mouse antibody against α-actin (A2228) and rabbit antibody against HA (H6908) were both obtained from Sigma-Aldrich. Sheep antibody against HIV-1 p24 was purchased from Biochrom AG. Rabbit antibodies directed against viral Tat (ab43014) and SRSF2 (ab28428) were provided by Abcam. Mouse antibody against phosphorylated SR proteins (1H4G7) was obtained from Invitrogen. Rabbit antibody against MS2 was provided by Tetracore (TC7004). For detection, we used a horseradish peroxidase (HRP)-conjugated anti-mouse antibody (NA931) from GE Healthcare, a HRP-conjugated anti-rabbit antibody (A6154) from Sigma-Aldrich and a HRP-conjugated anti-sheep antibody from Jackson Immunoresearch Laboratories Inc. For immunofluorescence analysis we used an anti-HA antibody (Sigma-Aldrich, clone HA-7) to detect the HA-tagged SRSF6 variants and an Alexa Fluor® 488 conjugated AffiniPure goat anti-mouse IgG (1:200; Jackson ImmunoResearch) as secondary antibody.

Whole-cell extracts (10⁶ cells per experimental point) were prepared using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) supplemented with protease inhibitors (Sigma-Aldrich). Proteins were separated using SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were incubated with a rabbit polyclonal antibody to HIV-1 Tat (ab43014, Abcam, Cambridge, UK) and a rabbit monoclonal antibody to β-actin (Cell Signaling Technology #4970, Danvers, MA, USA), which served as a loading control. HRP-conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz, CA, USA) was used as a secondary antibody. Protein levels were visualized using the ECL LumiGLO detection kit (Upstate, Biotechnology UBI).