Stage 15 animal caps were lysed in buffer containing 1% NP-40, 10 mM HEPES (pH 8.0), 150 mM NaCl, supplemented with cOmplete EDTA-free protease and PhosStop phosphatase inhibitor cocktail tablets (Roche). Using standard protocols, 20–75 µg of total protein was loaded per lane, separated by SDS-PAGE, and transferred to PVDF membrane. Membranes were blocked and stained as per antibody specifications (list of antibodies in supplementary material Table S1 ). Relative levels of target proteins were determined wrt YFP or Noggin using ImageJ (http://rsbweb.nih.gov/ij/ ), ImageLab, or Odyssey CLx system (LI-COR Biotechnology, Lincoln, Nebraska), and normalized to relative levels of β-actin.
Imagelab
Manufactured by LI COR
Sourced in China
The ImageLab is a lab equipment product designed for image analysis and quantification. It provides a platform for capturing, processing, and analyzing various types of images, including gels, blots, and microplates. The core function of the ImageLab is to enable users to acquire, measure, and interpret visual data from their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx system, by LI COR (1 mentions)
Imagej, by LI COR (1 mentions)
Phosstop phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
Enhanced chemiluminescence, by Vazyme (1 mentions)
Bca protein quantification kit, by Ipsen (1 mentions)
Erk1 2, by Abmart (1 mentions)
P erk1 2, by Abmart (1 mentions)
Ripa lysate, by Meilun (1 mentions)
Sds page gel, by Ipsen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Absin (1 mentions)
Mmp 9, by Absin (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Ab139690, by Abcam (1 mentions)
Ab50818, by Abcam (1 mentions)
Ab216773, by Abcam (1 mentions)
Ab128874, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
4 protocols using imagelab
1
Quantitative Western Blot Analysis of Embryonic Proteins
2
Western Blot Analysis of Protein Expression
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(1) After the cells in each group were washed with PBS for 3 times, RIPA lysate (Meilunbio, Dalian, China) was used to isolate total protein from HA-VSMCs, and extracted proteins were quantified by a BCA Protein Quantification Kit (Epizyme, Shanghai, China). (2) SDS-PAGE gel (Epizyme, Shanghai, China) electrophoresis was performed, and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) and blocked with 5% slim milk for 1 h. (3) The membranes were incubated overnight at 4 °C together with relevant primary antibodies and then incubated with corresponding secondary antibody for 2 h at room temperature. (4) The protein bands were visualized by enhanced chemiluminescence (Vazyme, Nanjing, China) and quantified by Image Lab (LI-COR). β-actin, PCNA and MMP-9 antibodies were purchased from Absin (Beijing, China), while Notch1, Jagged-1, ERK1/2 and p-ERK1/2 were from Abmart (Shanghai, China).
3
Western Blot Analysis of Striatal Proteins
4
Western Blot Analysis of Bromodomain Proteins
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Cells were seeded (HEK293: 1
× 105 cells/well) in 12-well plates. Following compound
treatment, cells were lysed on ice with RIPA lysis and extraction
buffer (Thermo Fisher Scientific, 89901) supplemented with protease
inhibitor cocktail (Merck, 11697498001) and Benzonase Nuclease (Sigma-Aldrich,
E1014). Protein concentration was determined using the BCA assay (Thermo
Fisher Scientific, 23225). Samples were then prepared and loaded onto
NuPAGE 4–12% Bis–Tris Midi gels (Thermo Fisher Scientific,
WG1403A) followed by the transfer of the proteins onto nitrocellulose
membranes (EMD Millipore). The membranes were blocked for 1 h prior
to incubation with the primary antibodies using 5% Milk TBST. Membranes
were probed for Brd2 (Abcam, Ab139690, 1:1000), Brd3 (Abcam, Ab50818,
1:4000), and Brd4 (Abcam, Ab128874, 1:1000). Following overnight incubation
with the primary antibodies at 4 °C, the membranes were incubated
with secondary antibodies (Anti-rabbit, Abcam AB216773, 1:5000 or
antimouse, Abcam AB216774, 1:5000) and hFAB Rhodamine Anti-Tubulin
Antibody (Bio-Rad, 12004165, 1:10 000) for 1 h and then imaged
with a Bio-Rad imager (LI-COR Biosciences). All Western blots were
analyzed for band intensities using Image Lab from Bio-Rad (LI-COR
Biosciences). The data extracted from these blots were then subsequently
plotted and analyzed using Prism (v. 8.2.1, GraphPad).
× 105 cells/well) in 12-well plates. Following compound
treatment, cells were lysed on ice with RIPA lysis and extraction
buffer (Thermo Fisher Scientific, 89901) supplemented with protease
inhibitor cocktail (Merck, 11697498001) and Benzonase Nuclease (Sigma-Aldrich,
E1014). Protein concentration was determined using the BCA assay (Thermo
Fisher Scientific, 23225). Samples were then prepared and loaded onto
NuPAGE 4–12% Bis–Tris Midi gels (Thermo Fisher Scientific,
WG1403A) followed by the transfer of the proteins onto nitrocellulose
membranes (EMD Millipore). The membranes were blocked for 1 h prior
to incubation with the primary antibodies using 5% Milk TBST. Membranes
were probed for Brd2 (Abcam, Ab139690, 1:1000), Brd3 (Abcam, Ab50818,
1:4000), and Brd4 (Abcam, Ab128874, 1:1000). Following overnight incubation
with the primary antibodies at 4 °C, the membranes were incubated
with secondary antibodies (Anti-rabbit, Abcam AB216773, 1:5000 or
antimouse, Abcam AB216774, 1:5000) and hFAB Rhodamine Anti-Tubulin
Antibody (Bio-Rad, 12004165, 1:10 000) for 1 h and then imaged
with a Bio-Rad imager (LI-COR Biosciences). All Western blots were
analyzed for band intensities using Image Lab from Bio-Rad (LI-COR
Biosciences). The data extracted from these blots were then subsequently
plotted and analyzed using Prism (v. 8.2.1, GraphPad).
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