Protein lysates were boiled in reducing sample buffer and resolved on 4–20% Criterion TGX polyacrylamide gels (Bio Rad). Proteins were transferred to nitrocellulose membranes and incubated for at least 1 h in blocking buffer (5% non-fat dry milk in 0.1% TBS-Tween). This step was followed by an overnight incubation at 4°C in primary antibody diluted in blocking buffer. Primary antibodies were detected using species-specific horse radish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch) followed by incubation with enhanced chemiluminescence substrate (ECL, Sigma). Chemiluminescence was detected by exposing blots to autoradiography film (Daigger). Immunoblots were quantified using ImageJ software. Primary antibodies and dilutions employed as they appear: ZC3H14, (1:6000) (15 (link)), Nuclear Pore complex NUP93 and NUP62, (1:5000), (414, Abcam, ab24609), eIF5, (1:5000) (Santa Cruz, sc-282), THOC1, (1:1000), (Bethyl, A302-839A), THOC2, (1:2000), (Bethyl, A303-630A), ALYREF, (1:1000), (ALY, Santa Cruz, sc-32311), HuR, (1:1000), (Santa Cruz, sc-5261), NPM1, (1:1000), (B23, Santa Cruz, sc-6013), THOC5, (1:1000) (Bethyl, A302-119A), HSP90, (1:1000), (Santa Cruz, sc-13119), and NXF1, (1:1000), (TAP, Santa Cruz, sc-32319).
Protocol detail
Western Blot Analysis of Nuclear Export Proteins
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Antibodies
Antibody
Autoradiography
Buffer
Chemiluminescence
Dilutions
Horse radish peroxidase
Hsp90
Immunoblots
Membranes
Milk
Nitrocellulose
Npm1
Nuclear pore complex
Nup93
Polyacrylamide gels
Proteins
Tween
Corresponding Organization : Emory University
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Variable analysis
independent variables
- Protein lysates were boiled in reducing sample buffer
- Protein expression levels of ZC3H14, Nuclear Pore complex NUP93 and NUP62, eIF5, THOC1, THOC2, ALYREF, HuR, NPM1, THOC5, HSP90, and NXF1
- Protein lysates were resolved on 4–20% Criterion TGX polyacrylamide gels (Bio Rad)
- Proteins were transferred to nitrocellulose membranes
- Membranes were incubated for at least 1 h in blocking buffer (5% non-fat dry milk in 0.1% TBS-Tween)
- Membranes were incubated overnight at 4°C in primary antibody diluted in blocking buffer
- Primary antibodies were detected using species-specific horse radish peroxidase (HRP) conjugated secondary antibodies (Jackson ImmunoResearch)
- Chemiluminescence was detected by exposing blots to autoradiography film (Daigger)
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