Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 1. ORF66 is essential in KSHV and required for late gene transcription 600

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques:

Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 3. The C-terminal domain of ORF66 is essential for protein-protein interactions 627

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques: Protein-Protein interactions

Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 5. The CxnC motifs in ORF66 are required for interaction with ORF34 652

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques:

Journal: FEBS letters

Article Title: Malonate induces the assembly of cytoplasmic stress granules.

doi: 10.1002/1873-3468.12049

Figure Lengend Snippet: Fig. 3. The association between eIF2a pathway and malonate-induced SG aggregation. (A) Total cell lysates from 100 mM malonate-treated or untreated HeLa cells for 1 h were subjected to SDS/PAGE and then blotted with rabbit anti-eIF2a, anti-Phoshpo-eIF2a (p-eIF2a) antibodies. (B) HeLa cells were transfected with the PERK siRNA, HRI siRNA, GCN2 siRNA, PKR siRNA, or scramble siRNA as control. After 48 h, the levels of PERK (a), HRI (b), GCN2 (c), and PKR (d) mRNA were analyzed by Q-PCR and normalized to GAPDH mRNA. Independent-Sample Student’s t-test was performed. Significant difference was indicated: # P < 0.05, ## P < 0.01. (C) After transfection with the above siRNAs, respectively, HeLa cells were treated with 100 mM malonate for 1 h or not. Western blotting assay was then performed to detect the phosphorylation level of eIF2a with indicated antibodies. (D) Band density was digitized using the IMAGE J software, and one-way analysis of variance (ANOVA) was performed. The level of p-eIF2a was normalized against eIF2a protein. Significant difference was indicated: # P < 0.05. (E) IF assay was performed to detect the formation of SGs (G3BP marker). Bar, 10 lm. (F) The number of SGs per cell was also measured and divided into four categories, including 0 ~ 10, 11 ~ 20, 21 ~ 30 and > 30, according to SG number per cell.

Article Snippet: HRI siRNA (sc-39052), GCN2 siRNA (sc-45644), PERK siRNA (sc-36213), PKR siRNA (sc-36263) and scramble siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (California, USA).

Techniques: SDS Page, Transfection, Control, Western Blot, Phospho-proteomics, Software, Marker

Journal: FEBS letters

Article Title: Malonate induces the assembly of cytoplasmic stress granules.

doi: 10.1002/1873-3468.12049

Figure Lengend Snippet: Fig. 4. The association between 4EBP1 pathway and malonate-induced SG aggregation. (A) Total cell lysates from 100 mM malonate-treated or untreated HeLa cells for 1 h were subjected to SDS/PAGE and then blotted with the rabbit anti-4EBP1, rabbit anti-Phoshpo-4EBP1 (p- 4EBP1), or anti-b-actin antibodies. (B) HeLa cells were transfected with the 4EBP1 siRNA, or scramble siRNA as control. Western blotting assay was then performed to detect the level of 4EBP1. (C) Band density was digitized using the IMAGE J software, and independent-sample Student’s t-test was performed. The level of 4EBP1 was normalized against b-actin protein. Significant difference was indicated: ## P < 0.01. (D) IF assay was performed to detect the formation of SGs (G3BP marker). Bar, 10 lm. (E) The number of SGs per cell was also measured and divided into four categories, including 0 ~ 10, 11 ~ 20, 21 ~ 30 and > 30, according to SG number per cell.

Article Snippet: HRI siRNA (sc-39052), GCN2 siRNA (sc-45644), PERK siRNA (sc-36213), PKR siRNA (sc-36263) and scramble siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (California, USA).

Techniques: SDS Page, Transfection, Control, Western Blot, Software, Marker

Journal: Communications Biology

Article Title: Characterization of exoribonuclease XRN1 as a cancer target and identification of adenosine-3’,5’-bisphosphate as a potent enzyme inhibitor

doi: 10.1038/s42003-025-08005-y

Figure Lengend Snippet: a XRN1, pPKR, total PKR and loading control western blot for NCI-H1650, NCI-H1703, NCI-H838, and NCI-H1944 cell lines transduced with non-targeting (NT), XRN1, or POL2RL sgRNA for 7 days. b RT-qPCR for Type I interferon genes, IFNA1 and IFNB1 , 7 days post CRISPR transduction (black bars: Non-targeting control; gray bars: XRN1 knockout). Error bars represent standard deviation of three replicates. c Proliferation of NCI-H838 cells (black bars: Non-targeting control; gray bars: XRN1 knockout) following 96 hours of stimulation with exogenous IFNβ, as assessed by a luminescent assay. Error bars represent standard deviation of three replicates. d Model for XRN1 in cancers with elevated dsRNA burden and rationale for monotherapy and combination with checkpoint inhibitors.

Article Snippet: Antibodies used were as follows: XRN1 (Cell Signaling Technology (CST) 70205; 1:1000), β-Actin (CST 3700; 1:2000), total PKR (R&D systems MAB1980; 1:500), phospho-Thr451 PKR (Abcam ab81303; 1:500), IRDye 800CW Goat anti-Rabbit (Li-Cor 926-32211; 1:20,000), and IRDye 680RD Goat anti-Mouse (Licor 926-68070; 1:20,000).

Techniques: Control, Western Blot, Transduction, Quantitative RT-PCR, CRISPR, Knock-Out, Standard Deviation, Luminescence Assay