Journal: PLoS ONE

Article Title: ATP hydrolytic activity of purified Spf1p correlate with micellar lipid fluidity and is dependent on conserved residues in transmembrane helix M1

doi: 10.1371/journal.pone.0274908

Figure Lengend Snippet: Specific activities reported in nmol/min/mg for purified and lipid/detergent reactivated Spf1p with the conditions for individual experiments shown. Star indicate activity measurements using radiolabeled ATP 32 , C12E10: Polyoxyethylene(10)dodecyl Ether, OG: n-Octyl-β-D-Glucopyranoside, PC/POPC: phosphatidylcholine and n.r.: not reported. For comparison polyamine stimulated activity of lipid activated ATP13A2 is ~150 nmol/min/mg [ 2 ] and for Ypk9p ~1.000 nmol/min/mg [ 18 ] both at 37⁰C, phosphatidylserine stimulated activity of the lipid flippase ATP8A1 is ~100.000 nmol/min/mg at 37⁰C [ 36 ], the activated state of the Pma1p H + ATPase is reported at ~12.000 nmol/min/mg at 30⁰C [ 37 ] and activity for SERCA and SPCA Ca 2+ ATPases are reported at ~12.000 nmol/min/mg and ~2500 nmol/min/mg respectively in proteoliposomes [ 38 ].

Article Snippet: Yeast polar lipid extracts (Avanti 190001) were dissolved in chloroform from the vial provided by the manufacturer and 1mg fractions were aliquoted into glass vials and stored at -200C under N 2 .

Techniques: Purification, Activity Assay, Comparison

Journal: Nature Communications

Article Title: The missense mutation Y65C in PQBP1 causes microcephaly and cognitive deficits through a combination of partial loss-of-function and gain-of-function effects

doi: 10.1038/s41467-025-68202-5

Figure Lengend Snippet: a Venn diagram delineating the overlapping distribution of differential binding proteins across parallel IP-MS datasets. n = 3 mice in each group in each independent experiment. b Top 10 enriched biological processes ranked by P -value. Analysis of the overlapping proteins from ( a ) using a one-sided hypergeometric test with Benjamini–Hochberg FDR correction. Numbers and the size of circles represent counts. c Heatmap visualization reveals differential expression patterns of enhanced binding proteins from ( a ). IBAQ intensities from duplicate IP-MS experiments were quantile-normalized and averaged. d Scatter plot depicting the genes with APA changes (FDR < 0.05) in E12.5 Pqbp1 Y65C/Y cortex. Blue: Transcripts with proximal PAS preference in Y65C. Red: Transcripts demonstrating distal PAS preference in Y65C. n = 3 mice in each group. e The genes with APA changes (FDR < 0.05) in E12.5 Pqbp1-cKO cortex. Blue: transcripts with proximal PAS preference in cKO compared to the control; Red: Transcripts demonstrating distal PAS preference in cKO. n = 3 mice in each group. f Venn diagram shows the overlapped targets between Pqbp1 Y65C/Y and Pqbp1-cKO . g The genes overlaping with known ESC self-renewal regulators. h Integrative Genomics Viewer tracks show the representative gene reads of Marcksl1 from RNA-seq. i qRT-PCR measures the ratios of transcripts with the extended 3′ UTR of Marcksl1 . The locations of primer sets used to amplify specific regions are marked (top). n = 3 mice in each group, p = 0.0329. j CoIP of PQBP1 and FIP1L1. The cortex of mice at E15.5 was immunoprecipitated with anti-PQBP1 antibodies. n = 3 biologically independent experiments, p = 0.0085. k , l Dual-luciferase reporter assays for Flag, Flag-PQBP1, and Flag-PQBP1 Y65C groups. The ratios of R/F (Renilla luciferase 480 nm/firefly luciferase 560 nm) were normalized to the Flag group. CMV, CMV promoter; R luc, Renilla luciferase gene; IRES, internal ribosomal entry site; F luc, firefly luciferase gene. n = 5 biologically independent experiments, Flag vs Flag PQBP1: p < 0.0001; Flag vs Flag PQBP1 Y65C : p = 0.0002. All quantification data are represented as mean ± SD. j Two-tailed unpaired Student’s t test. l Two-tailed one-way ANOVA with Tukey’s multiple comparisons test to adjust for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were PQBP1 (16264-1-AP, Proteintech, 1:1000 for use); RPS3 (ab128995, abcam, 1:1000 for use); RPL13 (ab134961, abcam, 1:1000 for use); FIP1L1 (83863-2-RR, Proteintech, 1:1000 for use); anti-α-tubulin (T9026, Sigma-Aldrich, 1:5000 for use); anti-actin (T0022, Affinity, 1:5000 for use).

Techniques: Binding Assay, Protein-Protein interactions, Quantitative Proteomics, Control, RNA Sequencing, Quantitative RT-PCR, Immunoprecipitation, Luciferase, Two Tailed Test