Tritc conjugated secondary antibody

For the detection of Collagen II, cells (1×10⁵ per well) were seeded in 6-well glass bottom plates. After the cells were treated, they were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for 15 min. Nonspecific binding sites were blocked with 1% BSA in PBS for 2 h. Then, the cells were treated with primary antibody specific to Notch-1(1:200, diluted in 1% BSA) overnight at 4 °C. Thereafter, the cells were incubated with TRITC-conjugated secondary antibody (Beyotime, China) for 1 h in the dark. DAPI (Beyotime, China) was used to stain nuclei before capturing images. The images were acquired using a fluorescence microscope (Nikon, Japan). The red fluorescence indicates Notch-1 expression, and the blue fluorescence indicates nuclei.

To show the interaction of TMEM106A with lipid raft, a plasmid expressing myc-tagged lipid raft marker CD230 was cotransfected into 293T cells with a plasmid expressing TMEM106A-EGFP. At 16 h posttransfection, cells were fixed for 1 h with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.2% Triton X-100. The cells were stained with anti-myc antibody, TRITC-conjugated secondary antibody and DAPI (Beyotime biotechnology), washed 3 times with PBS, and photographed using a Laser Confocal Microscope (Zeiss LSM700). To show the interaction of TMEM106A with gp160, a plasmid expressing TMEM106A-mCherry was cotransfected into 293T cells with a plasmid expressing gp160-EGFP. To show the interaction of TMEM106A with Gag, a plasmid expressing myc-tagged TMEM106A was cotransfected into 293T cells with pHIV-Gag-iGFP-ΔEnv, which expresses a Gag-GFP fusion protein (Hubner et al., 2007 (link); Micsenyi et al., 2013 (link)). At 16 h posttransfection, cells were fixed for 1 h with 4% paraformaldehyde, washed with PBS 3 times, and permeabilized with 0.2% Triton X-100. Myc-tagged TMEM106A was stained with anti-myc antibody, TRITC-conjugated secondary antibody and DAPI, washed 3 times with PBS, and photographed using a Laser Confocal Microscope (Zeiss LSM700).

For the detection of p65 nuclear translocation, cells (1 × 10⁵ per well) were seeded in six‐well glass‐bottomed plate. After the cells were treated, they were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X‐100 for 15 min. Nonspecific binding sites were blocked with 1% BSA in PBS for 2 h. Then, the cells were treated with primary antibody specific to p65 (ab16502; Abcam, 1 μg/mL) overnight at 4°C. Thereafter, the cells were incubated with TRITC‐conjugated secondary antibody (Beyotime, China) for 1 h at dark. DAPI (Beyotime, China) was used to stain nuclei before capturing images. The images were acquired using a fluorescence microscope (Nikon, Japan). The green fluorescence indicated p65 expression, and the blue fluorescence indicated nuclei.