For protein analysis, cells were lysed in T-PER buffer (Thermo Fisher Scientific 78510) plus phosphatase inhibitor cocktail (Cell Signaling Technology #5872S). Protein concentration was determined using Pierce BCA Protein Assay (Thermo Fisher Scientific 23225). Western blots were performed using 10% and 4–12% gradient Bis-Tris precast gels (NuPage) on Invitrogen XCell SureLock Mini-Cell modules. Gels were run using 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Invitrogen NP000202) and transferred onto nitrocellulose transfer membrane using an XCell II Blot Module or iBlot Dry Blotting System (Thermo Fisher Scientific). Antibody binding was visualized on CL-XPosure film using ECL SuperSignal West Extended Duration Substrate (both from Thermo Fisher Scientific) or using the Odyssey CLx Imaging System (LI-COR Biosciences). Antibodies used: ATF4 (Cell Signaling Technology #11815), BiP (CST #3177), CHOP (CST #2895), eIF2a (CST #9722), phospho-eIF2a (CST #3398), IRE1α (CST #3294), PERK (CST #3192), PERK p-T980 (CST #3179), Spliced XBP-1 (BioLegend #619502), actin (Sigma A5441 1:3000).
Spliced xbp1
Manufactured by BioLegend
Sourced in United Kingdom, United States, China
Spliced XBP1 is a gene expression marker that detects the spliced form of the X-box binding protein 1 (XBP1) transcript. XBP1 is a transcription factor that plays a crucial role in the unfolded protein response, which is activated in response to endoplasmic reticulum stress. The spliced form of XBP1 is a key indicator of this cellular stress response.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Total eif2α, by Cell Signaling Technology (3 mentions)
Phospho eif2α, by Cell Signaling Technology (2 mentions)
Western lightening plus ecl, by PerkinElmer (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Xcell surelock mini cell modules, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Grp78, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ncoa3, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Gcdca, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Tudca, by Merck Group (1 mentions)
6 protocols using spliced xbp1
1
Western Blot Analysis of UPR Proteins
2
Western Blot Analysis of UPR Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting procedures has been described previously.48 (link) The primary antibodies used were ATF6 (Abcam, Cambridge, UK, Cat# ab122897), spliced XBP1 (Biolegend, London, UK, Cat# 619502), PERK (Cell signalling, Cat# C33E10), GRP78 (Fisher Scientific Ireland Ltd, Cat# PA1-014A), phospho-eIF2α (Cell signalling, Cat# 9721), total eIF2α (Cell signalling, Cat# 9722) and or β-actin (Sigma, Cat# A-5060) overnight at 4 °C. The membrane was washed three times with PBS-0.05% Tween and further incubated in appropriate horseradish peroxidase-conjugated secondary antibody (Fisher Scientific Ireland Ltd) for 90 min. Signals were detected using Western Lightening Plus ECL (PERKin Elmer, Dublin, Ireland).
3
Western Blotting for ER Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting procedures has been described previously [46 (link)]. The primary antibodies used were NCOA3 (Cell signalling, Cat# 2126), ATF6 (Abcam, Cat# ab122897), spliced XBP1 (Biolegend, Cat# 619502), PERK (Cell signalling, Cat# C33E10), phospho-eIF2α (Cell signalling, Cat# 9721), total eIF2α (Cell signalling, Cat# 9722), ATF4 (Santa Cruz Biotechnology, Cat# sc-200), cleaved caspase-3 (Cell Signalling, Cat# 9661) and or β-Actin (Sigma, Cat# A-5060) overnight at 4°C. The membrane was washed 3 times with PBS-0.05% Tween or TBS-0.1% Tween (total-eIF2α and phospho-eIF2α) where appropriate and further incubated in appropriate horseradish peroxidase-conjugated secondary antibody (Pierce) for 2h at room temperature. Signals were detected using Western Lightening Plus ECL (PERKin Elmer).
4
Investigating ER Stress Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Metformin, GCDCA, and TUDCA were obtained from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were purchased from a variety of vendors: KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany), GADD153 (CHOP), CREB-2 (ATF4) (Santa Cruz, Santa Cruz, CA, USA), spliced XBP-1 (BioLegend, San Diego, CA, USA); IRE1α, phospho-JNK, PARP-1 (Cell signaling Technology, Danvers, MA, USA); Cyclooxynease-2 (Cayman Chemical, Ann Arbor, MI, USA); b-actin and α-tubulin (Sigma-Aldrich).
5
Western Blot Analysis of Stress Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were lysed on ice with cold RIPA buffer plus complete protease inhibitor cocktail (Roche Applied Science). Cell lysates were clarified by centrifugation at 12000 g for 10 min, and protein concentration was determined by the BCA Reagent. Lysates were separated on NuPAGE 4–12% Bis-Tris gel electrophoresis, proteins were then transferred to nitrocellulose membrane and immunoblotted with the following antibodies: ATF4 (Cell Signaling, 11815, 1:1000), PERK (Cell Signaling, 9956, 1:2000), GAPDH (Cell Signaling, 3683, 1:2000), p-eIF2α (Cell Signaling, 3597, 1:500), total eIF2α (Cell Signaling, 9722, 1:2000), IRE-1 (Cell Signaling, 3294, 1:1000), and spliced XBP-1 (Biolegend, 619501, 1:500). All immunoblots were visualized by enhanced chemiluminescene. Raw data of all western blots were included in Supplementary Figs S4 and S5 .
6
Western Blotting for UPR Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting procedures have been described previously. The nitrocellulose membranes were blocked with the specific blocking solution for 2 h at room temperature. The nitrocellulose membranes were then treated with specific primary antibodies, including PERK (Cell Signalling, Danvers, MA, USA, Cat #3192), ATF6 (Abcam, Hong Kong, China, Cat #ab122897), Spliced XBP1 (Bio Legend, San Diego, CA, USA, Cat #619501), c-MYC (Santa Cruz Biotechnology Cat # sc-40, Dallas, TX, USA) and β-Actin (Sigma, London, UK, Cat #A-5060) at 4 °C overnight. After washing three times with PBS/0.05%Tween solution, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. The membranes were then washed twice with PBS/0.05%Tween and once with PBS, and finally, the signals were detected using Western Lightening chemiluminescent substrate (PERKin Elmer, Groningen, The Netherlands, Cat #NEL104001EA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!