Mouse tissues were prepared as described (Dechat et al., 1998 (link); Fong et al., 2004 (link); Jung et al., 2012 (link)). Snap-frozen mouse tissues were pulverized with a chilled metal mortar and pestle, resuspended in ice-cold phosphate-buffered saline, and homogenized with a glass tissue grinder as previously described (Jung et al., 2013 (link)). The cell pellets were resuspended in urea buffer, sonicated, and centrifuged to remove cell debris. SDS–PAGE was performed on a 4–12% gradient polyacrylamide Bis-Tris gel, transferred to nitrocellulose membranes, and incubated with the following antibodies: a goat polyclonal antibody against lamin A/C (sc-6215; Santa Cruz Biotechnology, Santa Cruz, CA), a goat polyclonal antibody against lamin B1 (sc-6217; Santa Cruz Biotechnology), a mouse monoclonal antibody against lamin B2 (33-2100; Invitrogen), and a goat polyclonal antibody against actin (sc-1616; Santa Cruz Biotechnology). Binding of primary antibodies was assessed with infrared dye–conjugated secondary antibodies (Rockland Immunochemicals, Boyertown, PA) and quantified with an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE).
Infrared dye conjugated secondary antibodies
Manufactured by Rockland Immunochemicals
Sourced in United States
Infrared dye–conjugated secondary antibodies are laboratory reagents used for protein detection and quantification. These antibodies are designed to bind to primary antibodies and emit fluorescent signals in the infrared spectrum, which can be detected by specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Sc 6217, by Santa Cruz Biotechnology (1 mentions)
Sc 6215, by Santa Cruz Biotechnology (1 mentions)
Sc 1616, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Rhodamine red x, by Jackson ImmunoResearch (1 mentions)
α tubulin antibodies clone b 5 1 2, by Merck Group (1 mentions)
Odyssey device, by LI COR (1 mentions)
His tag, by Cell Signaling Technology (1 mentions)
Histone h2a, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Jetpei transfection reagent, by Polyplus Transfection (1 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Roche (1 mentions)
5 protocols using infrared dye conjugated secondary antibodies
1
Isolation and Western Blotting of Mouse Tissue Lamin Proteins
2
Comprehensive Antibody Database for Neuroscience Research
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The antibodies α-Cpx (Troy Littleton, MIT, Cambridge, MA; Huntwork and Littleton, 2007 (link)) and α-phospho-Mad (PS1; Peter ten Dijke, Leiden University Medical Center, Leiden, Netherlands; Dan Vasiliauskas, Susan Morton, Tom Jessell, and Ed Laufer, Columbia University Medical Center, New York, NY; Persson et al., 1998 (link)) have been described previously. α-Rab11 antibodies were obtained from BD Biosciences, San Jose, CA (clone 47; Khodosh et al., 2006 (link)), α-TDP-43 antibodies (10782-2-AP) from Proteintech, Rosemont, IL, and α-tubulin antibodies (clone B-5-1-2) from Sigma-Aldrich, St. Louis, MO. Rhodamine Red-X– and Alexa 647-conjugated α–horseradish peroxidase (HRP) antibodies were obtained from Jackson ImmunoResearch, West Grove, PA. α-Dlg (4F3), α-Futsch (22c10), α-Wit (23C7), α-actin (JLA20), α-eve (2B8), and α-synapsin (3C11) antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Secondary antibodies for imaging were conjugated to Dylight 488 or Rhodamine Red-X (Jackson ImmunoResearch). Immunoblots were imaged using infrared dye–conjugated secondary antibodies (Rockland Immunochemicals, Pottstown, PA) on a LI-COR Odyssey device.
3
Western Blot Analysis of Protein Expression
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Cells were washed twice with cold PBS, and total protein was extracted with RIPA lysis buffer (Beyotime Ins. Bio, China) with protease inhibitor PMSF (Sangon, China). The isolation of histone was performed as described previously [18 (link)]. Protein concentration was determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Samples containing equal amount of protein were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with primary antibodies against phosphorylated or total MSK1(1:500), c-Jun (1:500), His-tag (1:500), histone H2A (1:1000) (all from Cell Signaling Technology, Beverly, MA), Fra-1(1:500, Santa Cruz Biotechnology, CA) and EBV LMP1 (CS1-4) (1:1000, DAKO, Glostrup), then incubated with infrared-dye-conjugated secondary antibodies (1:10000, Rockland Immunochemicals, Gilbertsville, PA) for 1 h at room temperature. Protein bands were visualized by Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA) and quantified using the Quantity One software (Bio-Rad, USA).
4
ATF1 Phosphorylation Antibody Validation
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Antibodies against Pin1, ATF1 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA); against phosphorylated ATF1 (Ser63) was from Abcam, Inc (Cambridge, MA, USA). Infrared-dye-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbertsville, PA, USA). JetPEI transfection reagent was from Polyplus (llkirch, France). A polyclonal antibody against phosphorylated ATF1 (Thr184) was raised in a rabbit. Specificity of the antibody was confirmed by dot blotting and ELISA assay (Supplementary Information ,Supplementary Figure S4 ).
5
Quantitative Western Blot Analysis
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Western blotting was performed as previously described.[11 (link)] Briefly, total protein from heart homogenate was extracted by sodium dodecyl sulfate (SDS) lysis buffer containing a protease inhibitor cocktail and phosphatase inhibitors (Roche, Mannheim, Germany). The protein content was measured using a bicinchoninic acid assay. The equal amount of protein samples were separated using 10% SDS-polyacrylamide gel electrophoresis and then were transferred to nitrocellulose membranes which were incubated with the ATF3cc antibodies (1:500, Santa Cruz) and glyceraldehyde phosphate dehydrogenase (GAPDH) Abs (1:1000, Santa Cruz) at 4°C overnight and then with infrared dye-conjugated secondary antibodies (1:10,000; Rockland Immunochemicals) in Tris-buffered saline with Tween for 1 h at room temperature. Images were quantified using the Odyssey Infrared Imaging System.
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