Cells were treated with compound for 72 h before collection by centrifugation at 300g for 5 min. Cells were washed in 1× PBS and resuspended in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% Triton X-100, 30 μg μl−1 benzonase and fresh protease inhibitors. After incubation for 3 min at room temperature, SDS was added to a final concentration of 1%. Samples were boiled in SDS loading buffer before western blotting using the NuPAGE electrophoresis and transfer system (Invitrogen) and near-infrared detection for H3K36me2 (Abcam, ab9049, RRID: AB_1280939; 1:2,000), histone H3 (Abcam, ab10799, RRID: AB_470239; 1:5,000), α-tubulin (DSHB, E7, RRID: AB_528499; 1:5,000) and NSD2 (Abcam, ab75359, RRID: AB_1310816; 1:1,000). Immunoblots were imaged on a Li-Cor Odyssey CLx and quantified in Image Studio Lite v5.2.5 (Li-Cor Biosciences).
Nupage electrophoresis and transfer system
Manufactured by Thermo Fisher Scientific
The NuPAGE electrophoresis and transfer system is a laboratory equipment used for the separation and transfer of proteins via gel electrophoresis. The system includes the necessary components for performing these tasks, such as pre-cast polyacrylamide gels, buffers, and transfer modules.
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Lab products found in correlation
2 protocols using nupage electrophoresis and transfer system
1
Histone Methylation and NSD2 Analysis
2
Tubulin Acetylation Analysis in HEK293T Cells
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HEK293T cells were
treated for 24 h with 10 or 30 μM25 or 10 μM
Tubacin or DMSO control. Cell lysates were analyzed by Western blot
using the NuPAGE electrophoresis and transfer system (Invitrogen),
and proteins detected with primary antibodies anti-α tubulin-K40ac
(1:2000, Abcam, ab179484) and anti-α tubulin (1:2000, Abcam,
ab7291) and secondary antibodies IRDye 800CW (1:5000, ThermoFisher,
A32735) and IRDye 488CW (1:5000, ThermoFisher, A11029). Immunoblots
were imaged on a Li-Cor Odyssey CLx.
treated for 24 h with 10 or 30 μM
Tubacin or DMSO control. Cell lysates were analyzed by Western blot
using the NuPAGE electrophoresis and transfer system (Invitrogen),
and proteins detected with primary antibodies anti-α tubulin-K40ac
(1:2000, Abcam, ab179484) and anti-α tubulin (1:2000, Abcam,
ab7291) and secondary antibodies IRDye 800CW (1:5000, ThermoFisher,
A32735) and IRDye 488CW (1:5000, ThermoFisher, A11029). Immunoblots
were imaged on a Li-Cor Odyssey CLx.
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