Immunofluorescence staining was done as previously described (Laudermilch et al., 2016 (link); Rose et al., 2014 (link); Tsai et al., 2016 (link)). Briefly, cells were cultured on coverslips (VWR), fixed with 4% PFA in PBS, and permeabilized for 10 min with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 4% BSA (Sigma-Aldrich) in PBS for 10 min and incubated with the appropriate antibodies for 45 min. The following antibodies were used at a 1:500 concentration: anti–K48-Ub (AB_11213655, Millipore), anti-HA (AB_390919, Roche), anti-Flag (AB_259529, Sigma-Aldrich), and anti-VCP (ab11433, Abcam). After five washes, cells were blocked with 4% BSA and incubated with secondary antibodies conjugated to Alexa Fluor 488 (Life Technologies) or Alexa Fluor 568 (Life Technologies) for 45 min. Cells were washed with PBS, incubated in Hoechst 33342 (Life Technologies) for 5 min, and washed in PBS wash before being mounted onto slides using Fluoromount-G (Southern Biotech).
Standard wide field images were obtained on a Zeiss Axio Observer D1 microscope with a 63× oil immersion objective and an AxioCam MRm microscope camera using AxioVision 4.8 software (Zeiss). Samples were also imaged on a LSM 880 laser scanning confocal microscope (Zeiss) with a C Plan-Apochromat 63×/1.40 oil DIC M27 objective using ZEN 2.1 software (Zeiss).
Standard wide field images were obtained on a Zeiss Axio Observer D1 microscope with a 63× oil immersion objective and an AxioCam MRm microscope camera using AxioVision 4.8 software (Zeiss). Samples were also imaged on a LSM 880 laser scanning confocal microscope (Zeiss) with a C Plan-Apochromat 63×/1.40 oil DIC M27 objective using ZEN 2.1 software (Zeiss).
