Duolink kit

Wild type or GIGYF1/2 DKO cells were plated in 8 well slides (PEZGS0816, Sigma). Cells were washed with PBS and fixed for 20 min with 3.7% formaldehyde, washed twice in PBS and permeabilised for 5 min with PBS + 0.2% Triton and washed in PBS. Proximity Ligation Assay (PLA) was carried out using Duolink Kit (DUO92101) from Sigma following manufacturer instructions, except PLA probes were diluted 1:10. Antibodies used were: p97 (ab11433, Abcam) 1:300, GYGF2 (24790-1-AP, Proteintech) 1:300, UFD1 (10615-1-AP, Proteintech) 1:100. Images were obtained with a Leica SP5.

Proximity Ligation Assays (PLA). The mouse/rabbit red starter Duolink kit (Sigma-Aldrich) was used for this experiment. Human skin fibroblast cells were seeded at 3×10⁴ cells per well. The cells were fixed and permeabilized. After permeabilization the cells were incubated in the blocking buffer (provided with the kit) for 1hr at 37 °C in a humidified chamber. Next, cells were incubated with the primary antibodies diluted in the antibody diluents for 1hr at room temperature. Primary antibodies used were mouse anti-HP1α (1:2000; EMD), rabbit anti-yH2AX (1:50; Cell Signaling Technologies), rabbit anti-Suv39h1 (1:800; Abcam). Cells were then washed in Buffer A (supplied with the kit) 2 times for 5min each and incubated with the PLA probes for one hour at 37 °C in a humidified chamber. Cells were washed 2 times for 5min each in Buffer A before the ligation step at 37 °C for one hour in a humid chamber. Following 2 times of 2 min washes, cells were incubated with the amplification mix for 2hr at 37 °C in a darkened humidified chamber. Cells were washed with 1× Buffer B (supplied with the kit) 2 times for 10minutes followed by a 1min wash with 0.01× Buffer B before mounting on cover slips using the mounting media supplied with the kit.

For each well, 40,000 HeLa cells were seeded in 24-well plates, grown for 48 h, fixed with 4% paraformaldehyde and permeabilized with 0.05% (v/v) Triton X-100. PLA assays using the Duolink kit (Sigma Aldrich, St. Louis, MO, USA, DUO 9200) were have been described [11 (link)]. After blocking, cells were incubated with mouse anti-SQSTM1 and rabbit anti-VAPB and thereafter with the corresponding PLA probes. After ligation and amplification using the kit reagents, the cells were stained for SQSTM1 and VAPB and mounted with DAPI. Images were acquired using an LSM510 confocal laser scanning microscope with a 63×/1.4 oil immersion lens. In total, 250 cells from three independent experiments were analysed using CellProfiler [72 (link)]. An unpaired t-test was used for statistical analysis with a confidence interval of 95%.

Proximity ligation assays (PLAs) were performed using Duolink kit (Sigma-Aldrich, St-Louis, MI, USA) according to the manufacturer’s instructions. PLAs were performed on paraffin-embedded hMSG sections using rabbit anti-AQP5 (1:100; Millipore, Burlington, MA, USA) and mouse anti-ezrin (1:100; Thermo-Fisher Scientific, Waltham, MA, USA). PLAs were performed on methanol-fixed transfected NS-SV-AC cells using mouse anti-HA-tag (1:100; Proteintech, Rosemont, IL, USA) and rabbit anti-ezrin (1:200; Cell Signaling, Danvers, MA, USA). Negative controls were performed in the absence of one or both antibodies. Z-stack images were acquired using a confocal microscope (LSM-710) with an ×63/1.4 PlanApochromat lens (Zeiss, Oberkochen, Germany) and processed as previously described [12 (link)].

We performed a PLA to detect close interactions between Sec13 and Sec13 using a Duolink kit (Sigma-Aldrich, St. Louis, MO, USA) as previously described [69 (link)]. We used the mouse anti-Sec23 and rabbit anti-RFP antibodies to detect the complex containing Sec13 and Sec23, and mouse anti-RFP and rabbit anti-Sec23 antibodies to detect the complex containing Sec13 and Sec23. Negative control experiments were also performed to confirm that only anti-RFP antibody produced a few PLA-positive foci. Image acquisition was performed as described above.