rabbit polyclonal anti phospho perk thr980  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression

doi: 10.1038/s41467-019-09263-1

Figure Lengend Snippet: Increased Chop in CD8 + tumor-infiltrating T lymphocytes (TILs) correlates with poor survival in ovarian cancer. a Ddit3 mRNA levels in tumor-associated CD45 + CD8 + T cells (TILs) sorted from subcutaneous 3LL, EL-4, MCA-38, or B16 tumors and CD8 + T cells from the spleens of the same tumor-bearing mice (Tumor bearing) or tumor-free mice (Tumor free). Bar graphs show the mean ± s.e.m. ( n = 5 mice/group). b Chop expression in CD8 + TILs from B16 melanoma tumors (left) and 3LL tumors (right), compared with splenic CD8 + T cells from the corresponding tumor-bearing mice. Chop was detected by fluorescence-activated cell sorter and levels indicated by mean fluorescence intensity (MFI). Representative findings from four repeats. c CHOP in CD8 + TILs from ovarian carcinoma patients (Ovarian Ca tumor, n = 18) compared to peripheral blood CD8 + T cells from ovarian carcinoma patients (Ovarian Ca blood, n = 11) or healthy controls (Healthy blood, n = 6). d CHOP levels in autologous CD8 + TILs (Tumor) and peripheral blood CD8 + T cells (Blood) from ovarian carcinoma patients ( n = 7). e Representative image (scale 10 μm) showing CHOP expression in CD8 + TILs from ovarian carcinoma patients compared to CD8 + T cells from healthy ovarian tissues. Isotype (red) or CHOP (red, tested by clone 9C8 (left) or polyclonal antibody R-20 (right)), CD8 (green) and DAPI (blue) were detected by confocal microscopy. f Percentage of nuclear CHOP + cells (clone 9C8) among tumor-associated CD8 + T lymphocytes in a tissue microarray containing advanced ovarian carcinoma tissues ( n = 87) vs. normal ovarian tissues ( n = 12). g Overall survival in advanced ovarian tumor patients having increased frequency of nuclear CHOP in CD8 + TILs (CHOP high ) ( n = 52) vs. those having low frequency of nuclear CHOP in CD8 + TILs (CHOP low ) ( n = 29) (logrank 4.39, p = 0.0361 using Gehan–Breslow–Wilcoxon test; cutoff was established as described in the Methods section). Studies were developed using the anti-CHOP antibody clone 9C8. h Percentage of CD8 + TILs having nuclear CHOP (clone 9C8) in ovarian cancer patients that had optimal ( n = 59) vs. suboptimal ( n = 23) cytoreductive debulking surgery. Bar graphs represent mean value ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 were calculated using two-tailed unpaired Student’s t test

Article Snippet: The following antibodies were used for immunoblotting: Phospho-Perk (T980) (1:1000, 16F8 rabbit polyclonal, #3179), Perk (1:1000, C33E10 rabbit polyclonal, #3192), IREα (1:1000, 14C10 rabbit polyclonal, #3294), Atf4 (1:1000, D4B8 rabbit polyclonal, #11815), GCN2 (1:1000, rabbit polyclonal, #3302), Granzyme B (1:2000, rabbit polyclonal, #4275), and EOMES (1:1000, D8D1R rabbit polyclonal, #81493) antibodies were purchased from Cell Signaling Technology.

Techniques: Expressing, Fluorescence, Confocal Microscopy, Microarray, Two Tailed Test

Journal: Scientific Reports

Article Title: Hydrocortisone-induced parkin prevents dopaminergic cell death via CREB pathway in Parkinson’s disease model

doi: 10.1038/s41598-017-00614-w

Figure Lengend Snippet: Parkin inducers prevent HEK-293T cell death induced by oxidative stress. ( A ) Real time quantitative PCR quantification of relative parkin mRNA levels in HEK-293T cells treated by the top 15 compounds selected from luciferase assay (Fig. ). Their levels were normalized to that of GAPDH as an internal loading control ( n = 3). ( B ) Western blot analysis of parkin expression in HEK-293T cells treated with 10 µM of indicated compounds. DMSO was used as vehicle control (upper panel). Relative parkin protein levels in each compound-treated group were normalized to the level of β-actin. DMSO was used as a negative control for compound treatment ( n = 3 independent experiments per group). ( C ) Representative western blot images of PERK and phosphorylated PERK (pPERK) in HEK-293T cells treated with the indicated parkin inducing compounds (left panel). Relative pPERK levels were normalized to total PERK levels (right panel) as an indicator of ER stress ( n = 3 per group). ( D ) Viability of HEK-293T cells treated with indicated parkin inducing compounds and challenged with hydrogen peroxide (500 µM, 90 min) determined by trypan blue exclusion assay ( n = 3 per group). Quantified data are expressed as mean ± s.e.m. * P < 0.05, ** P < 0.01 and *** P < 0.001, unpaired two-tailed Student’s t test ( B ) or ANOVA test followed by Tukey post hoc analysis ( A , C , D ). Full blots (for cropped images in B , C ) are presented in Figure .

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against parkin (cat# 4211, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against pPERK (cat# 3179, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against PERK (cat# 5683, 1:5,000, Cell Signaling Technology), rabbit polyclonal antibody against CREB (Cat# 9197, 1:5,000, Cell Signaling Technology), and rabbit polyclonal antibody against AIMP2 (cat# 10424-1-AP, 1:5,000, Proteintech).

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Control, Western Blot, Expressing, Negative Control, Trypan Blue Exclusion Assay, Two Tailed Test