Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 1. Co-localization of RPS27A/MT2A/HMOX1/UBB and HSPC238 in 293T and SMMC7721 cells. 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag together with either pcDNA3.1-HMOX1-6xHis, pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. The co-localization of HSPC238 with (A) RPS27A, (B) MT2A, (C) HMOX1 and (D) UBB, was visualized on a confocal microscope with anti-His and anti-Flag antibodies. The blue fluorescence is the nuclei stained with DAPI, the green fluorescence is the His-tagged target protein and the red fluorescence is Flag-tagged HSPC238.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Microscopy, Fluorescence, Staining

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 2. HMOX1, MT2A, RPS27A and UBB co-immunoprecipitate with HSPC238. (A) 293T or SMMC7721 cells were transfected with pcDNA3.1, pcDNA3.1-HSPC238-Flag or pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for immunoblotting of His and Flag tag. (B) 293T or SMMC7721 cells were transfected with pcDNA3.1, pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total proteins were extracted for immunoblotting of the His tag. (C) 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag, or pcDNA3.1-HSPC238-Flag together with pcDNA3.1-HMOX1-6xHis pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total proteins were extracted for co-immu- noprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (IB) with anti-His tag, anti-HSPC238, anti-HMOX-1, anti-RPS27A, anti-UBB and anti-MT2A antibodies. (D) 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag or pcDNA3.1-HSPC238-Flag together with pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for co-immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (IB) with anti-His tag, HSPC238, HMOX-1, RPS27A, UBB and MT2A antibodies.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Western Blot, FLAG-tag, Immunoprecipitation

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 3. HSPC238 pull-down by HMOX-1, RPS27A, UBB and MT2A. (A) 293T or SMMC7721 cells were transfected with pcDNA3.1-HSPC238-Flag, alone or in combination with pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for simultaneous purification by nickel column. Three eluate samples with higher concentrations were subjected to western blotting. (B) 293T or SMMC7721 cells were transfected with pcDNA3.1-HSPC238-Flag, alone or in combina- tion with pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total protein was extracted for simultaneous purification by nickel column. The eluate sample with the highest concentration as well as cell lysates were subjected to western blotting.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Purification, Nickel Column, Western Blot, Concentration Assay

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 4. A possible mechanism of HSPC238 interaction with HMOX-1, MT2A, RPS27a and UBB.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques:

Journal: eLife

Article Title: Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice

doi: 10.7554/eLife.72182

Figure Lengend Snippet:

Article Snippet: Recombinant 6xHis-tagged mouse GRP78 was expressed in E. coli BL21 cells and purified on a HisTrap HP Nickel column (GE healthcare, Uppsala, Sweden).

Techniques: SYBR Green Assay, Sequencing, Recombinant, Plasmid Preparation, Software