Anti mcherry

Cells were collected using PBS and lysed for 10 min on ice using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS; Pierce, 89901) supplemented with proteinase inhibitor cocktail (Roche 1186153001) and PhosSTOP (Roche 04906845001). Samples were centrifuged for 20 min at 4°C at 14,000 rpm. 4X NuPAGE LDS sample buffer (Thermo Fisher Scientific NP0008) was added to the supernatant and samples were boiled for 5 min. Samples were run in 4–12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes using an iBlot 2 transfer device (Thermo Fisher Scientific). Membranes were blocked with Odyssey blocking buffer (LI-COR) and then incubated with primary antibodies. Following incubation with dye-labeled secondary antibodies, signals were visualized using an Odyssey Fc imaging system (LI-COR). Primary western blot antibodies were anti-β-actin (Santa Cruz Biotechnology sc-1616), anti-eIF2α (Santa Cruz Biotechnology sc-133132), anti-phospho-eIF2α (Cell Signaling 3597S), anti-mCherry (Abcam 167453), and anti-G3BP1 (BD biosciences 6111126). Secondary western blot antibodies were IRDye 800CW/680RD (LI-COR) used at a dilution of 1:15,000.

Human Flag-tagged altMiD51(WT) and altMiD51(LYR→AAA), and HA-tagged DrP1(K38A) were cloned into pcDNA3.1 (Invitrogen) using a Gibson assembly kit (New England Biolabs, E26115). The cDNA corresponding to human MiD51/MIEF1/SMCR7L transcript variant 1 (NM_019008) was also cloned into pcDNA3.1 by Gibson assembly. In this construct, altMiD51 and MiD51 were tagged with Flag and HA tags, respectively. MiD51^GFP and altMiD51^GFP were also cloned into pcDNA3.1 by Gibson assembly. For MiD51^GFP, a LAP tag (Hein et al., 2015 (link)) was inserted between MiD51 and GFP. gBlocks were purchased from IDT. Human altDDIT3^mCherry was cloned into pcDNA3.1 by Gibson assembly using coding sequence from transcript variant 1 (NM_004083.5) and mCherry coding sequence from pLenti-myc-GLUT4-mCherry (Addgene plasmid # 64049). Human DDIT3^GFP was also cloned into pcDNA3.1 by Gibson assembly using CCDS8943 sequence.
For immunofluorescence, primary antibodies were diluted as follow: anti-Flag (Sigma, F1804) 1/1000, anti-TOM20 (Abcam, ab186734) 1/500. For western blots, primary antibodies were diluted as follow: anti-Flag (Sigma, F1804) 1/1000, anti-HA (BioLegend, 901515) 1/500, anti-actin (Sigma, A5441) 1/10000, anti-Drp1 (BD Transduction Laboratories, 611112) 1/500, anti-GFP (Santa Cruz Biotechnology, sc-9996) 1/10000, anti-mCherry (Abcam, ab125096) 1/2000.

The anti-p62/SQSTM1 (Cat# 814802, dilution IFI 1/100; WB 1/1000 (v/v)) antibody was from BioLegend, the anti-LC3-B (Cat# 12741, dilution IFI 1/100 (v/v); WB 1/1000 (v/v)) and ATG5 (Cat# 12994, dilution IFI 1/100 (v/v); WB 1/1000 (v/v)) antibodies were from Cell Signaling Technology and the anti-HA (Cat# MMS-101R, dilution IFI 1/100 (v/v); WB 1/1000 (v/v), now available from BioLegend) antibody was from Covance. The anti-GFP (Cat# 029762, dilution WB 1/1000 (v/v)) antibody was from Chromotech. The anti-β-actin (Cat# PA1–183, dilution IFI 1/1000 (v/v)) antibody was from Thermo Fisher Scientific. The anti-mcherry (Cat# ab167453, dilution WB 1/5000 (v/v)) and anti-tubulin (Cat# ab18207, dilution WB 1/5000 v/v) antibodies were from Abcam.

When cells were treated with PRRSV or bergamottin for the indicated durations, cells were washed with PBS and harvested with a cell lysis buffer (Beyotime, Shanghai, China) on ice for at least 20 min. Whole-cell lysates were quantified using the BCA method (Beyotime, Shanghai, China). Quantified protein lysates were subjected to 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyl difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). After the electro-transfer, membranes were blocked with 5% skim milk (Sangon Biotech, Shanghai, China) in TBST (20 mM Tris-HCl PH8.0, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. Then, the membranes were cropped and incubated with indicated primary antibodies, including anti-PRRSV N (MEDIAN, Republic of Korea) antibody, anti-mCherry (Abcam, Cambridge, UK) antibody, and anti-GAPDH, p65, phosphorylated p65, IRF3, phosphorylated IRF3, phosphorylated IκBα (Cell Signaling Technology, Danvers, MA, USA) antibodies at a dilution of 1:1000 at 4 °C overnight. Among them, GAPDH served as an internal control. After rinsing with PBS three times, membranes were incubated with corresponding secondary antibodies at 1:5000 for 1 h. Finally, signals were visualized using an enhanced chemiluminescence reagent (NCM Biotech, Suzhou, China) on a Tanon 5200 system (Tanon, Shanghai, China).

Protein samples were resolved in a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes (GE Healthcare), blocked with 5% skimmed milk in TBST (Tris-buffered saline, 0.1% Tween 20) and probed with anti-GFP (Sigma-Aldrich), anti-mCherry (abcam) or anti-TET8. Then the signals were detected using the enhanced chemiluminescence method (GE Healthcare).