Fluorophore conjugated secondary antibody

In preparation for electrophoresis, each soluble fraction and remaining pellet fraction were prepared with 5X reducing loading dye for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. For Western blot, equal volumes of each sample were loaded into 12.5% polyacrylamide gels and separated by molecular weight. After separation, samples from each gel were transferred onto a nitrocellulose membrane. The gels were stained with Coomassie Blue and destained, revealing bands indicative of type I collagen α1 and α2 chains, and β-chains, indicating an α-chain dimer^{32 (link),40 (link)–44 (link)}. Membranes were blocked with Odyssey blocking buffer, (LI-COR Biosciences, Lincoln, NE) then incubated with primary rabbit anti-human catK polyclonal antibody (Protein Tech, Rosemont, IL) or goat anti-mouse catL monoclonal antibody (R&D Systems, Minneapolis, MN) followed by fluorophore conjugated secondary antibody (LI-COR Biosciences, Lincoln, NE) and imaged.

GFP (sc-9996), Raf (sc-133, sc-227) antibodies and purified recombinant MEK-1 (sc-4025) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AKT (AH01112) and Phospho-AKT (44623G) were from Invitrogen (Waltham, MA, USA). p42/44 MAPK (ERK1/2), pp44/42 MAPK (ERK T202/Y204), ppMEK (S217/221) and pAKT (S473) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Fluorophore-conjugated secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA). LY294002 was purchased from LC laboratories (Woburn, MA, USA). U0126 was purchased from Alfa Aesar (Ward Hill, MA, USA). Restriction endonucleases were obtained from New England Biolabs (Ipswich, MA, USA). pEGFP-C1 was from Clontech Laboratories (Mountain View, CA, USA). GFP-R-Ras constructs were made as described in Wurtzel et al.^{12 (link)} GFP-H-Ras G12V was a gift from K Svoboda (Addgene plasmid 18666). GFP-H-Ras (1–174)G12V- R-Ras (204–218) was generated from GFP-H-Ras G12V by PCR. GFP-RNex-H-Ras was generated by insertion of the first 26 amino acids of R-Ras from an R-Ras N-terminal domain construct originally described in Silver et al.,^{67 (link)} into the GFP-H-Ras background.

Viral and cell lysates were resuspended in sodium dodecyl sulfate (SDS) sample buffer and separated by electrophoresis on Bolt 4–12% Bis-Tris Plus gels (Life Technologies), blotted onto nitrocellulose membranes and probed overnight at 4°C with the following antibodies in Odyssey Blocking Buffer (LI-COR): mouse monoclonal anti-HIV p24 antibody (183-H12-5C, NIH AIDS reagents), mouse monoclonal anti-HIV integrase antibody (Bouyac-Bertoia et al., 2001 (link)), rabbit polyclonal anti-HIV integrase antibody raised in-house against Q44-LKGEAMHGQVD-C56 peptide and hence unlikely to be affected by the substitutions introduced into IN in this study, rabbit polyclonal anti-HIV-1 reverse transcriptase antibody (6195, NIH AIDS reagents), rabbit polyclonal anti-Vpr antibody (11836, NIH AIDS Reagents), rabbit polyclonal anti-MA antibody (4811, NIH AIDS Reagents). Membranes were probed with fluorophore-conjugated secondary antibodies (LI-COR) and scanned using an LI-COR Odyssey system. IN and CA levels in virions were quantified using Image Studio software (LI-COR). For analysis of the fates of core components in infected cells, antibody incubations were done using 5% non-fat dry milk. Membranes were probed with HRP-conjugated secondary antibodies and developed using SuperSignal West Femto reagent (Thermo-Fisher).

Immunoblot analysis was carried out as previously described^{29 (link)}. Briefly, cells (1 × 10⁶) were treated with PFK158 (concentration-dependent and time-dependent) and 40 μg of proteins were separated in SDS–PAGE (4–12.5% gradient gel) followed by electrotransfer onto nitrocellulose membrane, blocked with 3–5% TBS–BSA, probed overnight with primary antibodies (Supplementary information) at 4 °C and washed with 0.1% Tween-20-containing TBS. Immunocomplexes were identified with fluorophore-conjugated secondary antibodies (LI-COR). The membrane was washed and target proteins were identified by the LI-COR OdysseyFc Imaging System (Nebraska, USA).
For detection of the protein complex, the cell lysates containing 400 μg of protein were incubated with the anti-Rac1 antibody (1:100) overnight at 4 °C, and then 10 μl of 50% protein A-agarose beads were added and thoroughly mixed at 4 °C for 6 h. The immunoprecipitates were washed thrice with chilled PBS, collected and precipitated beads were loaded into the sample buffer, subjected to electrophoresis on 4–12.5% SDS–PAGE and blotted using an anti-Rab7 or anti-Calnexin or anti-Rac1 antibody.

The renal tissues were fixed with 4% paraformaldehyde made in PBS (pH 7.2), embedded in paraffin, and cutted into 5 μm sections. After 1 h blocking with appropriate 10% (v/v) normal serum at room temperature, the slides were incubated with primary antibodies of AQP1, AQP2, AQP3, and AQP4 (as above) at ratio of 1 : 100 overnight at 4°C. After washing three times, slides were incubated with fluorophore-conjugated secondary antibodies (LI-COR Biosciences Co., UK) at a ratio of 1 : 300 for 1 h at room temperature. The wavelength of the Licor antibody is 700 nm. Fluorescence signals were visualized and captured by using Cytation 5.

Whole cell lysates (50 μg of protein) were prepared from transfected HEK293T cells in 1% SDS buffered with HEPES plus 100 mM NaCl and complete protease inhibitor cocktail (Roche). The proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). Western blots were probed with rat anti-CD36 (Mab1955; R&D systems) and mouse anti-β-tubulin (Source Bioscience UK) primary antibodies, using 1:1,000 and 1:2,000 dilutions respectively, in blocking buffer (5% dried milk in PBS plus 0.1% Tween 20). Appropriate fluorophore-conjugated secondary antibodies were obtained from Licor and used at a dilution of 1:20,000, allowing detection of labelled proteins using the Odyssey infrared imaging system (LiCor).