Irdye 800cw donkey anti mouse igg secondary antibody

Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB). Fluorescent secondary antibodies used were Alexa Fluor 488 (Cat# A32766, Molecular Probes, Thermo Fisher Scientific 1:200 for IF) and 594 (Cat# A32754, Molecular Probes, Thermo Fisher Scientific, 1:200 for IF), IRDye^® 800CW Donkey anti-Mouse IgG Secondary Antibody (926-32212, LiCor Bioscience, Lincoln, NE, USA), and IRDye^® 680RD Donkey anti-Rabbit IgG Secondary Antibody (926-68073, LiCor Bioscience).

Western blots were performed as described previously (Ma and Mayr, 2018 (link)). Imaging was captured on the Odyssey CLx imaging system (Li-Cor). The antibodies used are mouse anti-α-tubulin (Sigma-Aldrich, T9026, RRID:AB_477593), mouse anti-mCherry (Abcam, ab125096, RRID:AB_11133266), rabbit anti-HuR (Millipore, 07-1735, RRID:AB_1977173), IRDye 680RD donkey anti-rabbit IgG secondary antibody (Li-COR Biosciences, 926-68073, RRID:AB_10954442), and IRDye 800CW donkey anti-mouse IgG secondary antibody (Li-COR Biosciences, 926-32212, RRID:AB_621847).

20 μg of cell protein was loaded for electrophoresis. Blots were incubated with HSP47 antibody (1:1000, #NBP1-97491, Novus Biologicals) or Sigma Receptor antibody (1:200, #sc-137075, Santa Cruz) overnight at 4 °C. After being washed three times, blots were incubated with IRDye 800CW donkey anti-mouse IgG secondary antibody (#925-32212, LiCor) at a 1:10,000 dilution for 1 h at RT. Blots were imaged using an Odyssey IR Imaging System. GAPDH was used an internal control.

2 µM SidH WT or SidH HM, 1 µM UBE2D2, 1 µM GST-Lubx, 0.3 µM E1, 12 µM ubiquitin and 2.5 mM ATP were incubated for 1 h at 30 °C. Samples were loaded onto 4–20% Tris glycine gradient gels (Biorad) and analysed by Coomassie staining or western blot using either Ubiquitin P4D1 (sc-8017, SantaCruz) or GST (sc-138, SantaCruz) antibody in a solution containing 5% BSA, 0.2% Tween20 and PBS. Following numerous washing steps with 0.2% Tween20 in PBS, blots were incubated with IRDye® 800CW Donkey anti-Mouse IgG Secondary Antibody (Li-Cor) for 1 h at room temperature and visualized by fluorescence. Following SDS-P AGE and coomassie staining, the gel streak corresponding to ubiquitinated SidH were excised and subjected to in-gel digestion with trypsin to generate peptides containing the Lys-ε-Gly-Gly (diGLY) remnant. The peptides were introduced into the Orbitrap Fusion Lumos (Thermo Scientific, SanJose) mass spectrometer. Data analysis was done with MSFragger v3.7^{41 (link)}. Carbamidomethyl (C) was set as fixed modification, Acetyl (Protein N-term), Oxidation (M) and GlyGly (K) as variable modifications.

The same protein lysates were used as above for the western blot experiment. 2 µl of protein lysates were pipetted on a nitrocellulose membrane for each sample. The membrane was then blocked in 5% BSA for 1 h followed by washing using PBST three times for 10 min each. Blot was incubated in primary antibody at 1:1000 dilution for RyR (MA3-916, Invitrogen) and GAPDH (ab37168, Abcam) for 1 h. The membrane was washed by PBST three times for 10 min each and labeled with IRDye^® 680RD donkey anti-mouse IgG secondary antibody and IRDye^® 800CW donkey anti-mouse IgG secondary antibody (LI-COR Biosciences) both at 1:10000 dilution for 1 h. Blots were washed before imaging. Image Studio Lite was used to analyze the dot blots. The presented blots derive from the same experiment and were processed in parallel.

Samples were loaded onto 4–20% Tris glycine gradient gels (Biorad) and analysed by western blot (WB) with RAD18 (NB100-61063, Bio-Techne Ltd), PCNA PC10 (sc-56, SantaCruz), Ubiquitin P4D1 (sc-8017, SantaCruz), MAGEA 6C1 (sc-20034, SantaCruz), HA-tag (C29F4, Cell Signalling), GFP (sc-9996, SantaCruz), or GAPDH (sc-32233, SantaCruz) antibody in a solution containing 5% BSA, 0.2% Tween20 and PBS. Following numerous washing steps with 0.2% Tween20 in PBS, blots were incubated with secondary antibodies for 1 h at room temperature and visualized by fluorescence. IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody (Li-Cor) was used as the secondary antibody to detect RAD18 and HA-tagged RAD18. IRDye® 800CW Donkey anti-Mouse IgG Secondary Antibody (Li-Cor) was used to detect MAGEA4, Ubiquitin, PCNA, GFP and GAPDH.

McCoy cells were infected with the transformants carrying aTc-inducible plasmids encoding C11orf83_6xH, Cls_TM-c11orf83_6xH, or OppB_TM-c11orf83_6xH. At 16 hpi, the constructs were induced with 2 or 10 nM aTc, and the infected cells were harvested at 24 hpi. The cells were resuspended with lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1X Halt protease inhibitor cocktail, 5 mM EDTA, 150 μM clasto-lactacystin β-lactone, and universal nuclease) and rotated for 20 min at 4°C at 10 rpm. The cell-debris and insoluble fraction were removed by centrifugation at 14,000 rpm for 30 min. The supernatant was removed to a new microcentrifuge tube, and the concentration was measured by EZQ protein assay kit (Thermo) following the manufacturer’s instructions. 100 μg crude extract was separated by 15% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Thermo). To detect six histidine tagged constructs, the membrane was blotted with mouse anti-6xH (Genscript, Piscataway, NJ) and IRDye 800CW Donkey anti-Mouse IgG Secondary Antibody (LICOR, Lincoln, NE). To detect MOMP, we used goat anti-MOMP (Meridian, Cincinnati, OH) and IRDye 680RD Donkey anti-Goat IgG Secondary Antibody (LICOR). The membrane was imaged using an Azure c600 imager system (Azure Biosystems, Dublin, CA).