tet on 3g system  (TaKaRa)

Journal: Scientific Reports

Article Title: Signal recognition particle-dependent secretome in humans

doi: 10.1038/s41598-026-35427-3

Figure Lengend Snippet: Relative protein abundance in HeLa cells and conditioned medium after SRP54 depletion. ( A ) Representative scan of the SDS-PAGE gels of radiolabeled proteins in SRP54-depleted (siSRP54) and control cells or medium 1, 2, and 4 h after metabolic labeling with EasyTag EXPRESS S protein labeling mix (PerkinElmer). See “Methods” for experimental details. ( B ) Graphs show the quantification of labeled proteins from ( A ) using Image J from three replicates. Average signal was used for the plot, standard deviation is shown. Statistical analysis was performed in GraphPad Prism using Two-way Anova with Sidak’s multiple comparisons ( p > 0.05, ns; p < 0.05, *). ( C , D ) Volcano plots of differentially abundant proteins determined by mass spectrometry in cells ( C ) and medium ( D ) after SRP54 depletion. Total of 6236 proteins were identified in cells and 1862 proteins in medium with a minimum exclusive peptide count of 2 and a protein false discovery rate (FDR) at 0.1%. Differentially abundant proteins with p < 0.05 and FC ≥ 1.2 decrease (log 2 FC ≤ − 0.263; 880 proteins in cells, 128 proteins in medium) or increase (log 2 FC ≥ 0.263; 581 proteins in cells and 171 in medium) are marked in red. Proteins that have not met statistical significance ( p > 0.05) or fold change are in green and blue, respectively; proteins that have not met both criteria are in dark gray. A few representative proteins from up- and down regulated groups are labeled. The relevant data to panels ( C ) and ( D ) are presented in Supplementary Files and .

Article Snippet: HeLa Tet-On cell line was from Clontech.

Techniques: Quantitative Proteomics, SDS Page, Control, Labeling, Standard Deviation, Mass Spectrometry

Journal: Scientific Reports

Article Title: Signal recognition particle-dependent secretome in humans

doi: 10.1038/s41598-026-35427-3

Figure Lengend Snippet: Identification and characterization of proteins with signal peptides and transmembrane domains in HeLa cells and medium after SRP54 knockdown. ( A ) Schematic diagram to identify affected and unaffected proteins in cells. Out of 6236 total proteins detected in cells, 1054 proteins were identified with either signal peptides (SPs) and/or transmembrane domains (TMDs). The annotations are from UniProtKB. Affected by SRP proteins have FC ≥ 1.2 decrease or increase (log 2 FC≤ − 0.263 and log 2 FC ≥ 0.263) that is statistically significant ( p < 0.05). 323 proteins with decreased abundancy (Down or SRP-dependent group, log 2 FC ≤ − 0.263) and 80 proteins with increased abundancy (Up group, log 2 FC ≥ 0.263) were detected in affected category. Unaffected proteins (No changes group or SRP-independent, 180 proteins total) have − 0.1 < log 2 FC < 0.1, or FC between 0.93 and 1.07 and a small coefficient of variation (CV) between replicates in the same group (CV < 10%). Proteins with small changes are determined by filtering p -value ( p < 0.05) and FC between 0.83 and 0.93 and FC between 1.07 and 1.2, or − 0.263 < log 2 FC ≤ − 0.1 and 0.1 ≤ log 2 FC < 0.263). Among those, 86 are in Down group (− 0.263 < log 2 FC ≤ − 0.1) and 66 are in Up group (0.1 ≤ log 2 FC < 0.263). Diagrams present analysis of proteins with SP only (blue), with TMD only (gray) or with both, SP and TMD (purple), in above categories. See Supplementary File for details of analysis. ( B ) Schematic diagram to identify affected and unaffected proteins in the medium. The same criteria as in panel A were used for analysis. Out of 1862 total proteins detected in medium, total 526 proteins were identified with either SPs and/or TMDs. Only one protein with a signal peptide was unaffected by SRP (Myeloid Derived Growth Factor, MYDGF). Within the affected group, 123 proteins with decreased abundancy (Down or SRP-dependent) and 5 proteins which increased abundancy (Up) were determined. The majority of SRP-dependent proteins (more than 90%) were with signal peptides. See Supplementary File for details of analysis. ( C ) Peptide intensity of LGALS3BP protein determined by mass spectrometry analysis in control and SRP54 knockdown cells. The protein contains signal peptide and identified as SRP-dependent. Two tailed paired t test was used for statistical analysis, p < 0.0001, ****. ( D ) The same as in ( C ) but in media samples. Statistical analysis was done as in ( C ), p < 0.001, ***. ( E ) Validation of mass spectrometry results for LGALS3BP protein by western blot. HeLa Tet-ON cells were transfected with non-target siRNA (Control) or siRNA against SRP54 (SRP54 KD), incubated for 48 h, and LGALS3BP was detected by western blot. Positions of protein markers in kDa are marked. ( F ) Analysis of mRNA level for LGALS3BP by Deep RNA sequencing from data obtained in previous study . Average transcript level of LGALS3BP mRNA was estimated based on three biological repeats. Two tailed paired t test was used for statistical analysis: p < 0.0001, ****. ( G ) Evaluation of the signal peptide (SP) grand average hydropathy index (hydrophobicity of each amino acid divided by the number of amino acids) was determined using the Kyte–Doolittle scale for all proteins categorized as SRP-independent (black line), Down (blue line), and Up (red line). These three categories were determined as described on panels ( A ) (for cell samples) and ( B ) (for media samples). Then, corresponding groups from cell and media were combined and duplicates were removed. Final numbers of proteins are 235 for Down, 61 for SRP-independent, and 35 for Up. Proteins with small changes in abundance were not included in the analysis. SPs were defined as published earlier , . ( H ) Same as ( G ) but only for amino acids contained in the signal peptide hydrophobic core (h-region). The SP regions were defined as published earlier , . ( I ) The net charge of the signal peptide n-region at pH = 7 based on the Lehninger pKa scale for all proteins that were analyzed in panels (G and H) as SRP-independent (black line, ), Down (blue line), or Up (red line). Proteins that did not have N-domain sequence determined were omitted from the analysis. ( J ) Total protein length distribution is defined as the number of amino acids on the X-axis. Relative count on the Y-axis indicates the proportion of proteins in each group with a specific number of amino acids. The same protein categories as for panels ( G – I ) were used.

Article Snippet: HeLa Tet-On cell line was from Clontech.

Techniques: Knockdown, Derivative Assay, Mass Spectrometry, Control, Two Tailed Test, Biomarker Discovery, Western Blot, Transfection, Incubation, RNA Sequencing, Sequencing

Journal: Scientific Reports

Article Title: Signal recognition particle-dependent secretome in humans

doi: 10.1038/s41598-026-35427-3

Figure Lengend Snippet: Cellular stress response and compensatory mechanisms associated with SRP54 depletion in human cells. ( A ) Effect of the SRP54 depletion on the cellular stress pathways: integrative stress response (ISR), Endoplasmic Reticulum Associated Protein Degradation (ERAD), unfolded protein response (UPR), Golgi-phagy, ER-phagy, UFMylation, and Rapid ER Stress-Induced Export (RESET). Volcano plot of stress protein abundances is shown, dashed lines corresponds to |log 2 FC| ≥ 0.263, and p < 0.05. The proteins with affected abundancies are labeled. Different dot colors correspond to specific pathway used in analysis. 45 proteins related to stress responses were detected in mass spec dataset and plotted on the graph. 18 proteins are decreased and 26 are not affected by SRP depletion. Only one protein, OS9, showed significant increase in abundancy. 3 proteins were detected in several pathways (example, HSPA5 labeled twice in the figure). See Supplementary File for details. ( B ) Functional enrichment analysis using gene ontology for cellular proteins with increased abundance in HeLa cells depleted of SRP54. Cnetplot is used to link up genes and top biological categories. Yellow dots represent categories. The size of the dot reflects the number of the affected proteins in this category. The range of fold change is presented in shades of blue. ( C ) Mass spec analysis shows relative levels of unique peptide intensities of ribosomal proteins RPS27 and RPS27L in SRP54-depleted cells and in control. Unpaired two-tailed t test was applied: *, p < 0.05; ***, p < 0.001, n = 3.

Article Snippet: HeLa Tet-On cell line was from Clontech.

Techniques: Labeling, Mass Spectrometry, Functional Assay, Control, Two Tailed Test