Donkey anti rabbit 800

4-[Aminoiminomethyl]-2,3-dihydro-1H-inden-1-diaminomethylene-hydrazone (SAM486A: also referred to as Sardomozide or CGP 48664) (0.4 µM in H₂O) was provided by Novartis. N1-guanyl-1,7-diamineheptane (GC7) (10 µM in H₂O) was purchased from LGC Biosearch Technologies. As recommended by the manufacturer, GC7 was used together in cell culture with 0.5 mM aminoguanidine to prevent destruction by monoamine oxidase (in H₂O). Deferiprone (DEF) (250 µM in H₂O) was purchased from Calbiochem. Ciclopirox (CPX) olamine (30 µM in H₂O), 2-difluoromethylornithine (DFMO) (200 µM in dimethyl sulfoxide [DMSO]), and N,N1-bis(2,3-butadienyl)-1,4-butanediamine (MDL) (50 µM in DMSO) were purchased from Sigma.
The following antibodies for immunoblots were used (the sources and dilutions shown in parentheses): rabbit anti-VP30 N-terminal region (prepared by GenScript; 1:5,000), rabbit anti-hypusine (Raghavendra Mirmira, Indiana University School of Medicine [35 (link)] and EMD Millipore; 1:1,000), mouse anti-GFP (Roche; 1:1,000), mouse anti-β-actin (Santa Cruz; 1:1,000), mouse anti-VP35 6c5 (Kerafast; 1:1,000), rabbit anti-NP (Integrated Biotechnologies; 1:2,000), rabbit anti-L (Integrated Biotechnologies; 1:1,000); IRDye secondary antibodies: donkey anti-mouse 680 and donkey anti-rabbit 800 (LI-COR Biosciences; 1:10,000).

Calu-3 cells were seeded at a density of 3 x 10⁵ cells/well in 12-well plates. Cells were infected with SARS-CoV-2 at an MOI of 1. Control cells were sham infected. Twelve hours post incubation, cells were transfected or treated with poly(I:C) or IFNβ, respectively for 6 h. Cell lysates were harvested for immunoblots and analyzed on reducing gels as mentioned previously (Banerjee et al., 2020b (link), 2021 (link)). Briefly, samples were denatured in a reducing sample buffer and analyzed on a reducing gel. Proteins were blotted from the gel onto polyvinylidene difluoride (PVDF) membranes (Immobilon, EMD Millipore) and detected using primary and secondary antibodies. Primary antibodies used were: 1:1000 mouse anti-SARS/SARS-CoV-2 N (ThermoFisher Scientific; Catalog number: MA5-29981; RRID: AB_2785780), 1:1000 rabbit anti-beta-actin (Abcam; Catalog number: ab8227; RRID: AB_2305186), and 2 μg/mL of mouse anti-ACE2 (R&D Systems; Catalog: MAB933; RRID: AB_2223153). Secondary antibodies used were: 1:5000 donkey anti-rabbit 800 (LI-COR Biosciences; Catalogue number: 926-32213; RRID: 621848) and 1:5000 goat anti-mouse 680 (LI-COR Biosciences; Catalogue number: 925-68070; RRID: AB_2651128). Blots were observed and imaged using Image Studio (LI-COR Biosciences) on the Odyssey CLx imaging system (LI-COR Biosciences).

The dilution used for the secondary antibodies was 1:1,000. Donkey anti-rabbit 800 (LI-COR Biosciences, 926-32213), donkey anti-rabbit 680 (LI-COR Biosciences, 926-68073) and goat anti-mouse 800 (LI-COR, 926-32210) for western blots. Donkey anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific, R37114, RRID: AB_2556542), donkey anti-guinea pig Alexa Fluor 488 (Jackson ImmunoResearch, 706-545-148, RRID: AB_2340472), donkey anti-rabbit Alexa Fluor 647 (Jackson ImmunoResearch, 711-605-152, RRID: AB_2492288) and donkey anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific, A-31570, RRID: AB_2536180).

Parasites from 9 mL of P. falciparum culture were isolated by saponin lysis, washed with PBS and resuspended in 1 x NuPAGE LDS sample buffer (Invitrogen). Proteins were separated by electrophoresis on 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. After blocking, membranes were probed with 1:2000 monoclonal mouse anti-FLAG M2 (Sigma) and 1:10,000 IRDye 680RD goat anti-mouse IgG (LiCor Bioscience, Lincoln, NE) for anti-FtsH1 immunoblots. For anti-PDF immunoblots, membranes were probed with 1:2000 rabbit monoclonal anti-MYC (Cell Signaling Technology 2278S, Danvers, MA), followed by 1:20,000 rabbit polyclonal anti-PfAldolase (Abcam ab207494, UK) and 1:10,000 donkey anti-rabbit 800 (LiCor Biosciences). Fluorescence antibody-bound proteins were detected with Odyssey Imager (LiCor Biosciences). When antibodies of the same species were used, membranes were probed and imaged sequentially. Immunoblots of FtsH-FLAG and PFD-myc were repeated in the laboratory >2 times.

Primary antibodies against the following proteins were used: CUL5 (ab184177; Abcam); RNF7 (ab181986; Abcam); UBE2F (ab185234; Abcam); Noxa (OP180; Millipore); Bim (ab32158; Abcam); Bid (ab32060; Abcam); Puma (ab9643; Abcam); Bad (ab62465; Abcam); Bik (ab52182; Abcam); Beclin (ab207612; Abcam); Bmf (ab9655; Abcam); MCL1 (D35A5 #5453; CST); Bcl-xL (54H6; CST); c-PARP (19F4 #9546; CST) Ubiquitin (P4D1 #3936; CST); HA (C29F4; CST); GAPDH (14C10 #2118; CST); p53 (DO-1 sc-126; Santa Cruz Biotechnology); HSP90 (sc-69703; Santa Cruz Biotechnology). For each protein antibody, manufacturer’s recommended dilutions were used. Mouse or rabbit immunoglobulin G was visualized at a 1:10,000 dilution: donkey anti-mouse 680 (925–68022; LI-COR); donkey anti-rabbit 680 (925–68023; LI-COR); donkey anti-mouse 800 (925–32212; LI-COR); donkey anti-rabbit 800 (925–32213; LI-COR). Blots were imaged on an Odyssey CLx Imaging System (LI-COR).

The following primary antibodies were used in this study: anti-FLAG (M2) and anti-myc (9E10) from Sigma-Aldrich, anti-GAPDH from Abcam (ab8245), anti-LC3B (GTX127375) and anti-C9orf72 (GTX119776) from GeneTex, anti-Cathepsin D (sc-6486), anti-Ulk1 (sc-33182) and anti-C9orf72 (sc-138763) from Santa Cruz Biotechnology, anti-C9orf72 (AP12928b) and anti-FIP200 (17250-1-AP) from Proteintech, anti-SMCR8 from Bethyl Laboratories, anti-WDR41 from Abgent, rat anti-CD68 from AbD Serotec, sheep anti-progranulin from R&D systems and anti-mouse LAMP1 (553792) from BD Biosciences. Anti-mouse prosaposin antibody was generated by Pocono Rabbit Farm and Laboratory and was previously characterized [55 (link)]. Anti-C9orf72-long isoform [52 (link)] was a gift from Dr. Janice Robertson (University of Toronto); anti-GFP antibody was a gift from Dr. Anthony Bretscher (Cornell University); and anti-GPP130 was a gift from Dr. William Brown (Cornell University). The following secondary antibodies were used: donkey anti-mouse 800 and donkey anti-rabbit 800 from LI-COR, AlexaFluor donkey anti-goat 680, donkey anti-rabbit 680, donkey anti-mouse 680, and donkey anti-rat 680 from Invitrogen, and donkey anti-mouse 568 from Biotium. Hoechst stain was obtained from Invitrogen. Brefeldin A (BFA) and nocodazole were obtained from Sigma-Aldrich and used at a final concentration of 300 μM and 20 mM, respectively.

To study the expression levels of each of the Myc-Borealin constructs, HeLa Kyoto cells were transfected in 12-well dishes as described above and solubilized after 36 h in 1× Laemmli buffer, boiled for 5 min, and analyzed by SDS-PAGE followed by Western blotting. The antibodies used for the immunoblot were rabbit anti-tubulin (1:10,000; ab18251; Abcam), mouse anti-myc (1:1,000; CSB-MA000041Mom, Cusabio), and mouse anti-Borealin (1:1,000; M147-3; MB). Secondary antibodies used were goat anti-mouse 680 and donkey anti-rabbit 800 (LI-COR) at 1:2,000 dilution. Immunoblots were imaged using the Odyssey CLx system.