Journal: Journal of Biological Chemistry

Article Title: The role of striated muscle Pik3r1 in glucose and protein metabolism following chronic glucocorticoid exposure

doi: 10.1016/j.jbc.2021.100395

Figure Lengend Snippet: Figure 6. Comparing signaling pathways that regulate protein synthesis in gastrocnemius muscle of WT and MKO mice. WT and MKO mice were treated with or without DEX (10 mg/kg body weight) for 1 week. At the end of treatment, mice were IP injected with insulin (1 unit per kg body weight) or PBS for 10 min, and gastrocnemius muscle was isolated and processed for ELISA to monitor the phosphorylation status of (A) p70S6K at Thr389, (B) 4E-BP1 at Thr37/Ser46 and (C) eIF2α at Ser52. n = 3 to 6, significance was denoted as *p ≤0.05. Error bars represent the SD. DEX, dexamethasone.

Article Snippet: All steps afterward were followed according to manufacturer’s instructions. eIF2α and phospho-eIF2α ELISA Relative total eIF2α and phosphorylated eIF2α levels were measured using the PathScan Phospho-eIF2α (Ser51) and total eIF2α Sandwich ELISA kits (Cell Signaling Technology, Catalog #7286 and Catalog #7952.)

Techniques: Protein-Protein interactions, Injection, Isolation, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Blood

Article Title: YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma

doi: 10.1182/blood.2019004147

Figure Lengend Snippet: 14-3-3ε interacts with and affects the mTORC1 signaling pathway. (A-B) Anti-FLAG antibody was used to pull down 14-3-3ε binding proteins in H929 KO cells expressing pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε). Cell lysate from H929 (FLAG-negative with YWHAE WT) cells was used as negative control. Pull-down products were subjected to mass spectrometry or western blot analysis. (A) Hallmark GSEA of 14-3-3ε interacting proteins detected by mass spectrometry analysis. (B) Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (C) Western blot analysis in H929 MM cells infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs was performed using the indicated mAbs, including 14-3-3ε mAb to confirm KD efficiency. (D) Western blot analysis in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε) was performed using the indicated mAbs, including 14-3-3ε to confirm addback efficiency. GAPDH was used as loading control. One representative blot of 2 is shown. (E) Pearson correlation coefficients between YWHAE and individual gene expression levels across all patients with MM were calculated; the resulting rank-ordered gene list was subjected to GSEA. GSEA false discovery rate (FDR)-q values and normalized enrichment scores for genes significantly correlated with YWHAE expression in primary MM cell RNA-seq data are shown in the graph. (F) GSEA-derived enrichment plots for mTORC1 and UPR pathways.

Article Snippet: The following antibodies were used: 14-3-3ε antibody (#9635; Cell Signaling Technology [CST]), mTOR Pathway Antibody Sampler Kit (#9964; CST), Phospho-TSC2 Antibody Sampler Kit (#8350; CST), 4E-BP Antibody Sampler Kit (#9955; CST), eIF2α (D7D3) XP® rabbit monoclonal antibody (mAb) (#5324; CST), Phospho-eIF2α (Ser51; D9G8) XP rabbit mAb (#3398; CST), and ANTI-FLAG M2 antibody (#F3165; MilliporeSigma).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Negative Control, Mass Spectrometry, Western Blot, Infection, RNA Sequencing Assay, Derivative Assay

Journal: Blood

Article Title: YWHAE/14-3-3ε expression impacts the protein load, contributing to proteasome inhibitor sensitivity in multiple myeloma

doi: 10.1182/blood.2019004147

Figure Lengend Snippet: Depletion of YWHAE inhibits translation initiation complex formation. (A-B) Western blot analysis in H929 (A) and KMS11 and JJN3 (B) MM cell lines infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs was performed using the indicated mAbs. (C) Western blot analysis in H929 (left) and KMS11 (right) KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε) was performed using the indicated mAbs, including 14-3-3ε to confirm addback efficiency. GAPDH was used as loading control. GAPDH ratio is shown (right). (D) m7GTP was used to pull down m7GTP binding proteins in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε). Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (E) Anti-FLAG antibody was used to pull down 14-3-3ε binding proteins in H929 KO cells expressing pLenti6-FLAG-YWHAE OE plasmid. Cell lysate from H929 with YWHAE KO cells expressing pLenti6-empty was used as negative control (empty). Pull-down products were subjected to western blot analysis and probed with the indicated antibodies. (F) Western blot analysis in H929 MM cells infected with either scrambled (pLKO.1) or two 14-3-3ε-targeted shRNAs (left), and in H929 KO cells expressing pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε; right). One representative blot of 2 is shown.

Article Snippet: The following antibodies were used: 14-3-3ε antibody (#9635; Cell Signaling Technology [CST]), mTOR Pathway Antibody Sampler Kit (#9964; CST), Phospho-TSC2 Antibody Sampler Kit (#8350; CST), 4E-BP Antibody Sampler Kit (#9955; CST), eIF2α (D7D3) XP® rabbit monoclonal antibody (mAb) (#5324; CST), Phospho-eIF2α (Ser51; D9G8) XP rabbit mAb (#3398; CST), and ANTI-FLAG M2 antibody (#F3165; MilliporeSigma).

Techniques: Western Blot, Infection, Expressing, Plasmid Preparation, Binding Assay, Negative Control