Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: Interaction between STRAP and three NOS memebers. A – C , coimmunoprecipitation analysis of STRAP interaction with iNOS ( A ), eNOS ( B ), or nNOS ( C ). HEK293 cells were cotransfected with FLAG-STRAP and iNOS, eNOS, or nNOS expression plasmids. Cell lysates were immunoprecipitated with anti-FLAG antibody or control mouse immunoglobulin G, followed by immunoblotting with anti-iNOS, anti-eNOS, or anti-nNOS antibodies. n = 2. D , detection of S-nitrosylated STRAP (SNO-STRAP) in HEK293 cells overexpressing iNOS, eNOS, or nNOS. Reaction without ascorbate (−Ascorbate) was included as a negative control. n = 3. E , expression levels of iNOS, eNOS, and nNOS in HEK293 cells. eNOS, endothelial nitric oxide synthase; HEK293, human embryonic kidney 293 cell line; iNOS, inducible nitric oxide synthase; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: Total lysates (2 mg) were incubated with freshly prepared 50 mM ascorbate and 50 μl thiopropyl-Sepharose (50% slurry) and rotated end over end in the dark for 4 h. The bound SNO proteins were sequentially washed with HEN, 1% SDS, and then 10% HEN, 0.1% SDS buffers; SNO proteins were eluted with 10% HEN, 1% SDS, 10% β-meracaptoethanol, and analyzed by SDS-PAGE and immunoblotting with mouse STRAP antibody (Proteintech, 66712) or mouse M2 FLAG antibody (Sigma).

Techniques: Expressing, Immunoprecipitation, Control, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: S-nitrosylation of STRAP abolishes its ability to suppress ASK1 activity and disrupts their interaction. A , S-nitrosylated STRAP (SNO-STRAP) after in vitro SNO donor Cys-NO treatment. B , ASK1 kinase activity in the presence of unmodified STRAP or SNO-STRAP. n = 5. C , interaction between STRAP and ASK1 following iNOS induction. FLAG-STRAP and Myc-ASK1 were coexpressed in HEK-iNOS cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-Myc antibody. “−” indicates the absence of doxycycline (Dox); “+” indicates the presence of Dox. n = 3. D , expression of iNOS and STRAP in HEK-iNOS cells transfected with no-targeting negative control siRNA or STRAP siRNA and induced by Dox. β-tubulin was used as a loading control. E , phosphorylation levels of MKK3 (Ser189) and p38 (Thr180/Tyr182) in HEK-iNOS cells transfected with control siRNA or STRAP siRNA. Cells were treated with H 2 O 2 , with or without Dox induction (“−” or “+”). n = 3. F and G , quantification of MKK3 phosphorylation and p38 phosphorylation levels in E . Phosphorylation levels of MKK3 and p38 (ImageJ signal intensities in E ) were first normalized to the corresponding pan-MKK3 and pan-p38 levels, respectively, and then were normalized to the si-control condition without Dox (−) and H 2 O 2 treatment (0 μM). All data in B , F , and G are presented as mean ± SD. Each data point represents one biological replicate. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05 and ∗∗ p < 0.01. ASK1, apoptosis signal–regulating kinase 1; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; MKK, mitogen-activated protein kinase kinase; NO, nitric oxide; SNO, S-nitrosolthiol; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: Total lysates (2 mg) were incubated with freshly prepared 50 mM ascorbate and 50 μl thiopropyl-Sepharose (50% slurry) and rotated end over end in the dark for 4 h. The bound SNO proteins were sequentially washed with HEN, 1% SDS, and then 10% HEN, 0.1% SDS buffers; SNO proteins were eluted with 10% HEN, 1% SDS, 10% β-meracaptoethanol, and analyzed by SDS-PAGE and immunoblotting with mouse STRAP antibody (Proteintech, 66712) or mouse M2 FLAG antibody (Sigma).

Techniques: Activity Assay, In Vitro, Immunoprecipitation, Expressing, Transfection, Negative Control, Control, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: S-nitrosylation of the scaffold protein STRAP enhances oxidative stress–induced apoptosis

doi: 10.1016/j.jbc.2026.111141

Figure Lengend Snippet: iNOS-mediated STRAP S-nitrosylation enhances sensitivity to H 2 O 2 -induced apoptosis. A , apoptosis in si-control and si-STRAP knockdown HEK-iNOS cells was measured using the RealTime-Glo Annexin V Apoptosis Assay. Cells were transfected with control or STRAP-targeting siRNA, induced with doxycycline (Dox) (“+”), and treated with H 2 O 2 . All samples were normalized to the average apoptotic cell percentage of si-control without Dox induction (−) and H 2 O 2 treatment (0 μM). B , cleavage of PARP and caspase 3 in si-control or si-STRAP knockdown HEK-iNOS cells following Dox induction (“+”) and H 2 O 2 treatment. Expression levels of iNOS and STRAP are shown in the upper panel . β-tubulin and GAPDH were used as a loading control. n = 3. C and D , quantification of cleaved PARP and cleaved caspase 3 levels in B . Cleaved PARP and cleaved caspase 3 levels (ImageJ signal intensities) were first normalized to the corresponding pre-PARP and pre-caspase 3 levels, respectively, and then normalized to the si-control condition without Dox (−) and H 2 O 2 treatment (0 μM). E , apoptosis in HEK293 cells expressing iNOS, eNOS, or nNOS was measured using the RealTime-Glo Annexin V Apoptosis Assay after H 2 O 2 treatment. F , cleavage of PARP and caspase 3 in HEK293 cells expressing iNOS, eNOS, or nNOS after H 2 O 2 treatment. Expression levels of iNOS, eNOS, and nNOS are shown in the lower panel . β-tubulin and GAPDH were used as a loading control. In B and F , spliced blots from the same gel for pre-caspase/cleaved caspase 3 or eNOS/iNOS/nNOS are indicated. The spliced blots were merged during enhanced chemiluminescence exposure for comparison. G and H , quantification of cleaved PARP and cleaved caspase 3 level in F . The cleaved PARP level and cleaved caspase 3 levels of all samples (ImageJ signal intensity) were first normalized to the corresponding pre-PARP and pre-caspase 3 levels, respectively, and then normalized to the levels of HEK293 cells without NOS expression (−) and H 2 O 2 treatment (0 μM). Results in A , C , D , G , and H are presented as mean ± SD. Each plot represents one biological repeat. One-way ANOVA with Tukey's post hoc test was used to detect the significance. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. eNOS, endothelial nitric oxide synthase; H 2 O 2 , hydrogen peroxide; HEK, human embryonic kidney cell line; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; PARP, poly(ADP-ribose) polymerase; STRAP, serine–threonine kinase receptor–associated protein.

Article Snippet: Total lysates (2 mg) were incubated with freshly prepared 50 mM ascorbate and 50 μl thiopropyl-Sepharose (50% slurry) and rotated end over end in the dark for 4 h. The bound SNO proteins were sequentially washed with HEN, 1% SDS, and then 10% HEN, 0.1% SDS buffers; SNO proteins were eluted with 10% HEN, 1% SDS, 10% β-meracaptoethanol, and analyzed by SDS-PAGE and immunoblotting with mouse STRAP antibody (Proteintech, 66712) or mouse M2 FLAG antibody (Sigma).

Techniques: Control, Knockdown, Apoptosis Assay, Transfection, Expressing, Comparison