Preparation of whole cell lysates, endoH treatment and Western blots were performed as previously described (Sha et al., 2014 (link); Sun et al., 2015 (link)). Antibodies used in this study were: Igμ-peroxidase (goat, 1:5,000) from Sigma; HSP90 (rabbit, 1:6,000), BiP (goat, 1:1,000) and α-Tubulin (mouse, 1:2000) from Santa Cruz; Sel1L (rabbit, 1:2,000) and OS9 (rabbit, 1:10,000) from Abcam; phospho-Syk and Syk (rabbit, 1:1000) from Cell Signaling; and Calnexin (rabbit, 1:8,000) from Assay Design. Hrd1-specific antibody (rabbit, 1:200) from Dr. Richard Wojcikiewicz at SUNY Upstate Medical University (Pearce et al., 2007 (link)); Antibodies for Bag6 (rabbit, 1:10,000) and H2A (rabbit, 1:10,000) were a kind gift from Dr. Yihong Ye (NIDDK). Band density was quantitated using the Image Lab software on the ChemiDOC XRS+ system (Bio-Rad). Protein levels were normalized to HSP90 and are presented as mean ± SEM unless otherwise specified.
Sel1l
Manufactured by Abcam
Sel1L is a protein that plays a role in the endoplasmic reticulum-associated degradation (ERAD) pathway. It functions as an adaptor protein, facilitating the recognition and transport of misfolded proteins from the endoplasmic reticulum to the proteasome for degradation.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Hsp90, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l hrp conjugate, by Bio-Rad (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Vinculin, by Merck Group (1 mentions)
Gapdh, by Novus Biologicals (1 mentions)
Calreticulin, by Cell Signaling Technology (1 mentions)
Ubiquitin, by Agilent Technologies (1 mentions)
Alexa fluor 594 f ab 2 fragment of goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Fam134b, by Proteintech (1 mentions)
Beclin 1, by Santa Cruz Biotechnology (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Rc dc assay, by Bio-Rad (1 mentions)
β actin, by Proteintech (1 mentions)
Gfp tag, by Proteintech (1 mentions)
Alpha tubulin, by Proteintech (1 mentions)
4 protocols using sel1l
1
Protein expression analysis by Western blot
2
Detailed Antibody Usage Protocol
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Primary antibodies: The following primary antibodies were used (antigen, dilution, vendor): Sel1L, 1:1000, Abcam ab78298; Actin, 1:4000, Sigma A5441; NPC1, 1:500 western, Abcam ab36983 (discontinued); NPC1, 1:200 imaging, Abcam ab134113; Flag, 1:1000, Sigma F1804; GP78, 1:1000, Cell Signaling 9590; Der-1, 1:1000, Cell Signaling 8897; HRD1, 1:1000, Cell Signaling. D302a; HA, 1:1000, Biolegend 901501; LC3, 1:1000 western, Novus NB600; LC3, 1:200 imaging, Gentex GTX17380; Beclin-1, 1:1000, Santa Cruz H-300; P97, 1:1000, Cell Signaling 2648; Ubiquitin, 1:500, Dako (discontinued); GAPDH, 1:5000, Novus NB600; Vinculin, 1:2000, Sigma V9131; FAM134B, 1:500, Abcam ab151755; FAM134B, 1:500, Proteintech 21537; p62, 1:1000, Sigma P0067; GBA, 1:1000, Sigma G4171; Calreticulin, 1:1000, Cell Signaling Technology 2891, GFP, 1:1000, Abcam ab13970. NPC1, 1:500, Abcam ab134113 was used for Figs. 3b and 5c , Supplementary Fig. S1b, c .
Secondary antibodies: The following secondary antibodies were used (antibody, dilution, vendor): Alexa Fluor 488 Goat anti-rabbit IgG (H+L), 1:500, Invitrogen A11008; Alexa Fluor 594 Fab’2 fragment of goat anti-mouse IgG (H+L), 1:500, Invitrogen A11020; Alexa Fluor 594 goat anti-chicken IgY (H+L), 1:1000, Invitrogen A11042; Goat anti-mouse IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6516; Goat anti-rabbit IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6515.
Secondary antibodies: The following secondary antibodies were used (antibody, dilution, vendor): Alexa Fluor 488 Goat anti-rabbit IgG (H+L), 1:500, Invitrogen A11008; Alexa Fluor 594 Fab’2 fragment of goat anti-mouse IgG (H+L), 1:500, Invitrogen A11020; Alexa Fluor 594 goat anti-chicken IgY (H+L), 1:1000, Invitrogen A11042; Goat anti-mouse IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6516; Goat anti-rabbit IgG (H+L)-HRP conjugate, 1:2000, Bio-Rad 170-6515.
3
ER Stress Protein Quantification
4
Immunoblotting Assay for Cellular Stress Markers
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Mammalian cells were lysed in Radioimmunoprecipitation assay buffer (RIPA) buffer with the addition of protease (Roche cOmplete), and phosphatase inhibitors (GB Sciences). Protein levels of lysates were determined using the BioRad DC/RC assay. Equal amounts of protein lysate were boiled with SDS load buffer, and equal amounts of protein were loaded. Immunoblotting was performed with the following antibodies: GAPDH (Proteintech, Cat: 60004-1-lg), ATF6 (Proteintech, Cat: 60004-1-lg), beta-Actin (Proteintech, Cat: 20536-1-AP), alpha-Tubulin (Proteintech, Cat: 66031-1-Ig), Sel1L (Abcam, Cat: ab78298), GFP tag (Proteintech, Cat: 66002-1-Ig), BiP (Proteintech, Cat: 11587-1-AP), ATF4 (Cell Signaling Technology, Cat: 11815S) .
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