Ab151558

The Duolink^® PLA (Merck) was used to detect close proximity of endogenous ALIX with CHMP4, Rab11, TSG101, KIF5B or KLC1. The assay is based on oligonucleotide-conjugated PLA probes, containing secondary antibodies directed against primary antibodies against the proteins of interest. Annealing of the probes occurs when the target proteins are in close proximity (< 40 nm), which then initiates the amplification. The amplicons can be detected by fluorescence microscopy in a quantifiable manner. For this assay, Hela K cells were seeded on coverslips in six-well plates and the PLA experiments were performed the next day with subconfluent cells. Cells were washed, fixed and permeabilized as described in the section above. Antibodies against ALIX (1:100, BioLegend #634502), CHMP4B (1:500, [16 (link)]), Rab11 (1:100, Invitrogen #71-5300), TSG101 (1:100, Sigma #HPA006161), KIF5B (1:100, Abcam #ab151558), KLC1 (1:100, Santa Cruz #sc25735) and tubulin (1:200, Cytoskeleton #ATN02) were used as primary antibodies, and the assays were performed as described in the manufacturer’s manual. Slides were mounted with ProLong Glass (Invitrogen) and samples were observed by fluorescence microscopy with a Nikon ECLIPSE Ti2 spinning-disk microscope (number of experiments: ALIX + CHMP4B = 4, ALIX + KIF5B = 4, ALIX + KLC1 = 4, ALIX + Rab11 = 2, ALIX + TSG101 = 2).

The following primary antibodies were used for Western blotting (WB) and/or immunofluorescence (IF): anti-β-actin (Sigma-Aldrich #A5316), anti-ALIX and anti-CHMP4B [16 (link)], anti-CEP55 (Abnova #H00055165-A01), anti-KIF5B (Abcam #ab151558), anti-KLC1 (1:100, Santa Cruz #sc25735), anti-GAPDH (Abcam #ab9484), and only for IF anti-ALIX (Bio Legend #634502), anti-RacGAP1 (Abcam #ab2270), and anti-tubulin (Sigma-Aldrich #T5168). Secondary antibodies included anti-mouse, anti-rabbit, and anti-goat Alexa Fluor 488 (Jackson ImmunoResearch), Alexa Fluor 555 (Molecular Probes), Alexa Fluor 568 (Molecular Probes), Alexa Fluor 647 (Jackson ImmunoResearch), and DyLight649 (Jackson ImmunoResearch). Methanol-free 16% paraformaldehyde (PFA) was from Thermo Scientific, SiR700-tubulin from Spirochrome and nocodazole was purchased from Merck.