Journal: Retrovirology

Article Title: Wip1 and p53 contribute to HTLV-1 Tax-induced tumorigenesis

doi: 10.1186/1742-4690-9-114

Figure Lengend Snippet: Analysis of Wip1 mRNA expression in Tax-expressing and Tax-non-expressing cells. ( A ) Total RNAs from HTLV-1–transformed MT-2, MT4, C8166 T-cell lines and HTLV-1-negative CD4 + control T-cell lines (Jurkat, CEM, and H9) were extracted and reverse transcribed. The cDNAs were used for real-time RT-PCR analyses of Wip1 , Tax , and GAPDH (internal standard) transcripts. The mRNA relative expression levels of Wip1 and Tax mRNA were determined and normalized as multiples of the GAPDH mRNA. The columns represent the average results from 3 experiments; the error bars are mean errors. ( B ) Real-time RT-PCR analyses of Wip1 and GAPDH (internal control) transcripts were performed in p53 −/− HCT116, ( C ) p53 +/+ HCT116 and ( D ) HeLa cells after transfection with a control vector or a Tax-expression vector. To detect Tax protein, immunoblots were stained using Tax and α-tubulin specific monoclonal antibodies. Tubulin was used as a loading control.

Article Snippet: Human T cell lines MT2, MT4, C8166, Jurkat, A301, CEM, and H9 were maintained in RPMI 1640 with 10% FBS.

Techniques: Expressing, Transformation Assay, Control, Reverse Transcription, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Staining, Bioprocessing

Journal:

Article Title: Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1 ▿

doi: 10.1128/JVI.01469-06

Figure Lengend Snippet: Cytoskeletal inhibitors reduce cell-cell spread, as measured by qPCR. Equal numbers of JurkatLAI cells and uninfected target cells were mixed and incubated for 1, 3, and 6 h prior to cell lysis and purification of total cellular DNA. qPCR using pol primers was performed to measure de novo viral DNA synthesis. Data were normalized to HSA and are shown as ratios of HIV-1 DNA to HSA DNA. The data obtained with A2.01 target cells were used as baseline HIV-1 DNA synthesis at the different time points and were subtracted from the signal with A3.01 target cells. (A) JurkatLAI cells were either left untreated or incubated with Q4120 or AMD3100 prior to being mixed with A3.01 target cells to inhibit Env-CD4 or Env-CXCR4 interactions, respectively. (B) JurkatLAI cells were either left untreated or incubated with an inhibitor of actin remodeling (latrunculin, cytochalasin D, or jasplakinolide) prior to being washed and mixed with A3.01 cells. (C) JurkatLAI cells were either left untreated or incubated with an inhibitor of microtubule remodeling (colchicine or nocodazole). Bars represent the means for triplicate samples plus 1 SD.

Article Snippet: The CD4 + CXCR4 + Jurkat CE6.1 T-cell line (American Type Culture Collection) and the A3.01 T-cell line (Center for AIDS Reagents [CFAR], United Kingdom) were maintained in suspension cell growth medium (RPMI 1640 [Gibco BRL] supplemented with streptomycin [100 μg/ml], penicillin [100 U/ml], and 10% fetal calf serum [FCS]).

Techniques: Incubation, Lysis, Purification, DNA Synthesis

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: The Ppp1r15b R658C variant leads to persistent ISR activation and disrupt protein synthesis in the brain of Ppp1r15b R658C mice. (A) Sanger sequencing confirms the generation of Ppp1r15b R658C mice carrying the Ppp1r15b R658C variant. (B) Comparison of brain size between WT ( n = 6) and Ppp1r15b R658C ( n = 6) mice ( t = 7.82, P < 0.0001; two-tailed Student’s t -test). (C) Representative Western blot (top) and quantification (bottom) of PPP1R15B protein levels in hippocampal extracts from WT ( n = 4) and Ppp1r15b R658C mice ( n = 4) [ t = 1.42, P = 0.21]. (D) Immunoprecipitation of WT PPP1R15B and mutant PPP1R15B R658C using an GFP antibody followed by Western blots of endogenous PP1 and eIF2 (left). A schematic of the WT and mutant phosphatase complex (right). (E) Representative Western blot (top) and quantification (bottom) of eIF2-P levels in hippocampal extracts from WT ( n = 4) and Ppp1r15b R658C mice ( n = 5) [ t = 3.82 , P < 0.001]. (F) Schematic of polysome profiling sedimentation. After ultracentrifugation, sub-polysomes (40S, 60S, and 80S) and polysomes are separated based on their size. ( G-H ) Averaged polysome profile traces ( G ) and quantification ( H ) of polysome/sub-polysome ratio in the brain of WT and Ppp1r15b R658C mice ( n = 6 per group, t = 3.19 , P < 0.01). ( I-J ) Detection of puromycin incorporation into nascent peptides using an anti-puromycin antibody. A representative Western blot ( I ) and quantification ( J ) in hippocampal extract from WT ( n = 4) and Ppp1r15b R658C mice ( n = 3) [ t = 4.73, P < 0.01]. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Variant Assay, Activation Assay, Sequencing, Comparison, Two Tailed Test, Western Blot, Immunoprecipitation, Mutagenesis, Sedimentation

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Persistent ISR activation cause deficits in long-term synaptic plasticity and memory in Ppp1r15b R658C mice. (A) Schematic of the contextual fear conditioning paradigm. (B) Long-term contextual fear memory in WT ( n = 23) and Ppp1r15b R658C ( n = 18) mice ( F 3,52 = 60.76, t = 5.73, P < 0.0001). (C) L-LTP induced by four trains of high frequency stimulation (HFS, 4 x 100 Hz) in WT ( n = 8) and Ppp1r15b R658C ( n = 12) mice (at 250 min: t = 4.14, P < 0.001). (D) Representative Western blot (top) and quantification (bottom) of eIF2-P levels in hippocampal extracts from WT ( n = 7), Ppp1r15b R658C mice ( n = 9) and Ppp1r15b R658C -Eif2s1 S/A ( n = 3) [ F 2,16 = 1.86, WT vs. Ppp1r15b R658C : t = 4.25, P < 0.01; Ppp1r15b R658C vs. Ppp1r15b R658C -Eif2s1 S/A : t = 3.60, P < 0.01; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 0.39, P > 0.99). (E) Long-term contextual fear memory in WT ( n = 7), Ppp1r15b R658C ( n = 13) and Ppp1r15b R658C - Eif2s1 S/A mice ( n = 8) [ F 2,50 = 5.58; WT vs. Ppp1r15b R658C : t = 4.31 , P < 0.001 ; Ppp1r15b R658C vs. Ppp1r15b R658C - Eif2s1 S/A : t = 5.12 , P < 0.0001; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 0.54 , P > 0.99). (F) L-LTP induced by four trains of high frequency stimulation (HFS, 4 x 100 Hz) in WT ( n = 9) and Ppp1r15b R658C ( n = 5) mice and Ppp1r15b R658C - Eif2s1 S/A ( n = 8) [at 250 min: F 2,19 = 8.69; WT vs. Ppp1r15b R658C : t = 3.18 , P < 0.01; Ppp1r15b R658C vs. Ppp1r15b R658C - Eif2s1 S/A : t = 4.09, P < 0.001; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 1.15, P = 0.264). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Protein alignment across diverse kingdoms of life reveals DP71L as a potent pan-ISR inhibitor. (A) Schematic of protein alignment showing PP1- and eIF2-binding motifs across various species. Consensus PP1- and eIF2-binding motif sequences are shown. (B) Overview of ISR kinases and the mechanism by which DP71L represses the ISR. ( C-E ) Cells expressing either GFP or GFP-DP71L were treated with the specific chemical inducer for each ISR kinase. Representative Western blot (top) and quantification (bottom) of eIF2-P levels upon activation of HRI ( C , F 3,16 = 7.14, n = 5 per group; GFP+Vehicle vs. GFP+Oligo: t = 4.22, P < 0.01; GFP-DP71L+Vehicle vs. GFP-DP71L+Oligo: t = 0.98, P > 0.99; GFP+Oligo vs. GFP-DP71L+Oligo: t = 4.17, P < 0.01), PKR [ D , n = 3-4 per group, F 3,10 = 9.80; GFP+Vehicle vs. GFP+Poly(I:C): t = 4.16, P < 0.05; GFP-DP71L+Vehicle vs. GFP-DP71L+Poly(I:C): t = 1.15, P > 0.99; GFP+Poly(I:C) vs. GFP- DP71L+Poly(I:C): t = 6.13, P < 0.001], PERK ( E, n = 3-6 per group, F 3,15 = 1.90, GFP+Vehicle vs. GFP+Tg: t = 5.52, P < 0.0001; GFP-DP71L+Vehicle vs. GFP-DP71L+Tg: t = 2.90, P = 0.06; GFP+Tg vs. GFP-DP71L+Tg: t = 5.99, P < 0.001) and GCN2 ( F, n = 3 per group, F 3,8 = 0.67; GFP+Vehicle vs. GFP+Ner: t = 6.29, P < 0.01; GFP-DP71L+Vehicle vs. GFP-DP71L+Ner: t = 2.20, P < 0.35; GFP+Ner vs. GFP-DP71L+Ner: t = 9.27, P < 0.001). Tg = thapsigargin, Oligo = oliogomycin, Ner = neratinib, Poly:IC = polyinosinic-polycytidylic acid: a synthetic double strand RNA. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Binding Assay, Expressing, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Molecular dissection of DP71L function. (A) Schematic comparison of DP71L and ΔPPP1R15B, highlighting their PP1- and eIF2-binding motifs. (B) Representative Western blot of eIF2-P levels following expression of either GFP-DP71L or GFP- ΔPPP1R15B. (C) Immunoprecipitation of GFP-DP71L or GFP-ΔPPP1R15B using a GFP antibody, followed by Western blot against endogenous PP1 or eIF2. (D) Schematic of the different GFP-DP71L/GFP-ΔPPP1R15B chimeras. (E) Immunoprecipitation of the different chimeras with a GFP antibody, followed by Western blotting for endogenous PP1. (F) Schematic of various GFP-DP71L and GFP-ΔPPP1R15B chimeras. (G) Immunoprecipitation of the different chimeras using a GFP antibody, followed by Western blotting for endogenous PP1.

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Dissection, Comparison, Binding Assay, Western Blot, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Structural characterization of DP71L function. (A) A 3.1 Å electron density map of the PP1•DP71L•eIF2α•G-actin complex (left), and the same map rotated 90°. DP71L is colored blue, PP1 is orange, eIF2α is yellow, and G-actin is copper. (B) Zoomed view of the linker region of DP71L and PPP1R15B, which was modeled into the cryo-EM map (green). (C) Structural modeling a DP71L mutant (4X) predicts an increase the size of the bulge and disruption of the hydrogen bonding, likely leading to decreased binding affinity for PP1. (D) Immunoprecipitation of WT GFP-DP71L and 4X GFP-DP71L with an GFP antibody, followed by Western blotting for endogenous PP1. (E) Structural modeling a mutant ΔPPP1R15B (3X) predicts a decrease in the size of the bulge and the formation of a new hydrogen bond, potentially enhancing binding to PP1. (F) Immunoprecipitation of WT ΔPPP1R15B and 3X ΔPPP1R15B with an GFP antibody, followed by Western blotting for endogenous PP1.

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Disruption, Binding Assay, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

doi: 10.1038/s41598-020-79379-8

Figure Lengend Snippet: Upregulation of 5′-TOP mRNA translation following loss of ARF. ( a ) Immunoblot analysis showing increased protein abundance of PABP, TPT1, RPL23A, EEF2 and RPL22. ( b ) Quantitation of blots in panel ( a ). Total protein was used for normalization. Mean ± SD, n = 6. ( c ) qPCR shows no change in mRNA expression of 5′-TOP mRNAs. Mean ± SD, n = 4. ( d ) The average fold change of TE from two ribosome profiling replicates is shown for PABPC1, TPT1, RPL23A, EEF2 and RPL22. ( e ) Schematic of the luciferase reporters used in panels ( f , g ). ( f ) Luciferase activity of a 5′-TOP reporter is increased in Arf −/− MEFs (Arf-KO). Luciferase activity was normalized to firefly luciferase transfection control and set relative to WT. Mean ± SD, n = 3. ( g ) qPCR shows no increase in mRNA expression of the 5′-TOP reporter. Normalized to firefly luciferase transfection control. Mean ± SD, n = 3. *, p-value < 0.05, ** p -value < 0.01; two-tailed t-test with Bonferoni correction.

Article Snippet: The blots were then probed with the appropriate primary antibodies: Abcam PABP (ab21060); Bethyl—GAPDH (A300-641A), LARP1 (A302-087A), EEF2 (A301-688A), RPL23A (A303-932A-T); Cell Signaling Technologies—TPT1(5128), p-MTOR (2971), p-S6 (2215S), p-S6K (9205S), p-4EBP1 (2855S), eIF4G1 (2858), p53 (2524S), eIF4E (9742S), 4EBP1 (9452S); Santa Cruz Biotechnology—ARF (sc-32748), RPL22 (sc-136413), University of Iowa, DSHB—puromycin.

Techniques: Western Blot, Quantitative Proteomics, Quantitation Assay, Expressing, Luciferase, Activity Assay, Transfection, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

doi: 10.1038/s41598-020-79379-8

Figure Lengend Snippet: Upregulation of 5′-TOP mRNAs in p53 −/− MEFs. p53 fl/fl MEFs were transduced with control AAV-LacZ or AAV-Cre to knockout p53 . ( a ) Polysome profiling showing increased polysome peaks following knockout of p53 (Cre). ( b ) Immunoblot analysis showing protein abundance of 5′-TOP encoded proteins (TPT1, RPL22, RPL23A, EEF2 and PABPC1) and 5′-TOP regulators (eIF4G1, LARP1, eIF4E and mTORC1) following knockout of p53 (AAV-Cre). ( c ) Luciferase activity of a 5′-TOP reporter is increased in p53 −/− MEFs. Luciferase activity was normalized to firefly luciferase transfection control and set relative to LacZ control. Mean ± SD, n = 3. ( d ) qPCR shows no increase in mRNA expression of the 5′-TOP reporter. Normalized to firefly luciferase transfection control. Mean ± SD, n = 3. ( e ) Quantitation of blots in panel ( b ) for TPT1, RPL22, RPL23A, EEF2 and PABPC1. Total protein was used for normalization. Mean ± SD, n = 3–7. ( f ) qPCR shows no change in mRNA expression of 5′-TOP mRNAs following knockout of p53 . Mean ± SD, n = 6. ( g ) Quantitation of blot in ( b ) and qPCR for LARP1. Mean ± SD, n = 7(protein), 6(RNA). ( h ) Quantitation of blot in ( b ) and qPCR for eIF4G1. Mean ± SD, n = 6(protein), 6(RNA). ( i ) Quantitation of blot in ( b ) and for mTORC1 pathway. Mean ± SD, n = 4. *, **, *** p -value < 0.05, < 0.01, < 0.001; two-tailed t-test with Bonferoni correction.

Article Snippet: The blots were then probed with the appropriate primary antibodies: Abcam PABP (ab21060); Bethyl—GAPDH (A300-641A), LARP1 (A302-087A), EEF2 (A301-688A), RPL23A (A303-932A-T); Cell Signaling Technologies—TPT1(5128), p-MTOR (2971), p-S6 (2215S), p-S6K (9205S), p-4EBP1 (2855S), eIF4G1 (2858), p53 (2524S), eIF4E (9742S), 4EBP1 (9452S); Santa Cruz Biotechnology—ARF (sc-32748), RPL22 (sc-136413), University of Iowa, DSHB—puromycin.

Techniques: Transduction, Control, Knock-Out, Western Blot, Quantitative Proteomics, Luciferase, Activity Assay, Transfection, Expressing, Quantitation Assay, Two Tailed Test