Journal: bioRxiv

Article Title: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

doi: 10.1101/2025.03.17.643278

Figure Lengend Snippet: RIF1-L phospho-SPKF peptide binds to tandem BRCT domain of BRCA1 in vitro (A) (B) Fluorescence polarisation analysis of binding affinities between BRCA1-BRCT domain and RIF1-L phospho-peptide with indicated treatment or mutations. (C) Crystal structure of RIF1-L phospho-peptide (stick presentation) in complex with BRCA1-BRCT domain (space-filling presentation), solved by X-ray diffraction. The phosphorus atom is represented in orange. Residues on RIF1-L phospho-peptide are labelled with pink text (S2265,K2267,F2268,K2269). Residue on BRCA-BRCT domain is labelled with green text (E1698). (D) Model of RIF1-L interaction with BRCA1. In RIF1-L protein presentation, purple bards indicate PP1-interacting motifs; pink box indicates Exon 31; red bar indicates the ‘S 2265 PKF’ motif. RIF1-L phosphoS 2265 PKF motif binds to the C-terminal tandem BRCT domains of BRCA1.

Article Snippet: Primary antibodies used for western blotting include anti-RIF1 (Bethyl Laboratories, A300-568A); anti-RIF1-L-Phospho-S2265 (amsbio, custom); anti-BRCA1 (Santa Cruz Biotechnology, sc-6954); anti-BARD1 (Bethyl Laboratories, A300-263A); anti-53BP1 (Novus Biologicals, NB100-94); anti-PP1α (Santa Cruz Biotechnology, sc-271762); anti-Tubulin (Santa Cruz Biotechnology, sc-53030); anti-FLAG (Sigma-Aldrich, F1804); anti-GFP (Chromotek, 3h9); anti-RFP (Chromotek, 5f8).

Techniques: In Vitro, Fluorescence, Binding Assay, Residue

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: 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: Translational Psychiatry

Article Title: Electrophysiology-based screening identifies neuronal HtrA serine peptidase 2 (HTRA2) as a synaptic plasticity regulator participating in tauopathy

doi: 10.1038/s41398-025-03227-4

Figure Lengend Snippet: A In the T maze test, spontaneous alteration percentage was studied for comparison. In the Morris water maze test, the escape latency during a six-day training was recorded for comparison for every day ( B ). On the seventh day, the time spent for the first entry into the target quadrant ( C ) and the number of platform crossings ( D ) were recorded for comparison (WT-NC = 10 mice, WT-HTRA2 = 10 mice, PS19-NC = 9 mice, and PS19-HTRA2 = 10 mice for all behavioral tests). E LTP recordings in the hippocampal CA1 region and quantification of the mean fEPSP slope in the last 10 min of recordings ( n = 8 slices from 4 mice per group). F Representative Western blotting of tau and phosphorylated (p-) tau in the hippocampal lysates of treated mice. Comparison of levels of total tau and phosphorylated tau at sites S202/T205, T181, and S396 ( n = 5 per group). G Representative Western blotting of tau kinases and phosphatases in the hippocampal lysates of treated mice. Comparison of levels of PP2B-Aα, PP1α, PP2A-α, phosphorylated GSK3β, CDK5, and P25 ( n = 5 per group). A significant difference was determined using one-way ANOVA with Tukey’s post hoc analysis ( A , C , D , E , G ), two-way ANOVA with Tukey’s post hoc analysis ( B ) and an unpaired t -test ( F ). Data represent means ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Antibodies used included: anti-NeuN (94403S, 1:1000), anti-β-actin (8457S, 1:10000), anti-GluN1 (5704S, 1:1000), anti-PSD93 (19046S, 1:1000), anti-p35/25 (2680, 1:1000), anti-PSD95 (3450S, 1:1000), anti-tau pS181 (12885S, 1:1000), and anti-SYN1 (5297S, 1:1000) from Cell Signaling Technology; anti-GFAP (16825-1-AP, 1:1000), anti-HTRA2 (15775-1-AP, 1:1000), anti-GluN2A (19953-1-AP, 1:1000), anti-GluN2B (19954-1-AP, 1:1000), anti-GSK3β (51065-1-AP, 1:1000), anti-GluA2 (11994-1-AP, 1:1000), anti-pT216-GSK3β (51065-1-AP, 1:1000), and anti-flag (66008-4-lg, 1:1000) from Proteintech; anti-PP2B-Aα (sc-17808, 1:1000) and anti-CDK5 (sc-173, 1:1000) from Santa Cruz Biotechnology; anti-PP1α (43-8100, 1:1000) and HRP-conjugated secondary antibodies (31460/31430, 1:5000) from Thermo Fisher Scientific; anti-Iba1 (016-20001, 1:500) from Wako; anti-tau pS202/T205 (MN1020, 1:500), anti-tau pS396 (44752 G, 1:1000), and anti-Total tau (Tau5, AHB0042, 1:1000) from Invitrogen; anti-Synaptophysin (S5768, 1:2000) from Sigma-Aldrich; anti-GluA1 (MAB2263, 1:1000) from Millipore; anti-GAPDH (AB0037, 1:5000) from Abways; and anti-PP2A-α (A6702, 1:1000) from ABclonal.

Techniques: Comparison, Western Blot