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New England Biolabs paired end multiplexed sequencing library
Paired End Multiplexed Sequencing Library, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/end+sequencing+libraries/Endonuclease+III/pmc12698818-116-24-28
Average 99 stars, based on 3489 article reviews

paired end multiplexed sequencing library - by Bioz Stars, 2026-09

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Article Title: A Bivalent Molecular Glue Linking Lysine Acetyltransferases to Oncogene-induced Cell Death
Article Snippet: IPs and inputs were reverse-crosslinked in TE/0.5% SDS/0.5 μg/μL proteinase K for 55 °C /3 hours then 65 °C /18 hours, then DNA was purified using a PCR cleanup spin column (Takara #74609). .. Paired-end sequencing libraries were constructed using an NEBNext Ultra II DNA kit (E7645S) and indexed (NEB E7335). .. Library size distributions were confirmed using an Agilent Bioanalyzer and High Sensitivity DNA reagents (Agilent 5067) and concentrations determined by qPCR.

Article Title: Unraveling the genetic basis of post-infancy diagnosed sensorineural hearing loss using whole exome sequencing.
Article Snippet: Objective: To clarify the genetic causes and the mutation spectrum of post-infancy diagnosed sensorineural hearing loss in the Chinese population.. Methods: We enrolled patients with post-infancy diagnosed bilateral sensorineural hearing loss (onset age between 1 and 60 years) at the Eye & ENT Hospital of Fudan University from November 2018 to October 2022.. Whole-exome-sequencing (WES) was performed to elucidate the genetic etiology of these patients.

Article Title: A case of Smith-Magenis syndrome with skin manifestations caused by a novel locus mutation in the RAI1 gene.
Article Snippet: We fragmented 1 to 3lg of genomic DNA, which was extracted from each sample, to an average size of 180 base pairs (bp) using a Bioruptor sonicator (Diagenode, Li ege, Belgium). .. Paired-end sequencing libraries were then prepared using a DNA sampleprep reagent set 1 (New England Biolabs UK Ltd., Hitchin, UK). .. Library preparations, including end repair, adapter ligation, and polymerase chain reaction enrichment, were performed following Illumina (San Diego, CA,USA) protocols.

Article Title: Genome-Wide Characterization of a Carbon Ion Beam-Induced Soybean Mutant Population Reveals Extensive Genetic Variation for Trait Improvement.
Article Snippet: DNA quality and concentration were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA). .. Paired-end sequencing libraries were constructed using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) following manufacturer protocols with minor optimizations for soybean genomic DNA. .. One microgram of genomic DNA was fragmented to 350–400 bp average size using Covaris S220 focused-ultrasonicator with optimized parameters to achieve uniform size distribution.

Article Title: Genetic Insights into the 'Sandwich Fusion' Subtype of Klippel-Feil Syndrome: Novel FGFR2 Mutations Identified by 21 cases of Whole-Exome Sequencing
Article Snippet: Preparation of DNA Library Blood samples from all patients were collected and forwarded to Beijing MyGenomics Inc., where genomic DNA (1–3 μg) was extracted from each specimen and fragmented to an average size of 180 bp using a Bioruptor sonicator (Diagenode, U.S.). .. Paired-end sequencing libraries were then prepared following standard protocols with NEBNext (Illumina, U.S.). ..

Article Title: Genome-Wide Characterization of a Carbon Ion Beam-Induced Soybean Mutant Population Reveals Extensive Genetic Variation for Trait Improvement
Article Snippet: DNA quality and concentration were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA). .. Paired-end sequencing libraries were constructed using the NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) following manufacturer protocols with minor optimizations for soybean genomic DNA. .. One microgram of genomic DNA was fragmented to 350–400 bp average size using Covaris S220 focused-ultrasonicator with optimized parameters to achieve uniform size distribution.

Article Title: Data on whole genome resequencing of selected Malaysian rice accessions with opposing response to salinity stress
Article Snippet: The DNA samples were sent to the sequencing service provider at the Apical Scientific Sdn Bhd. (Selangor, Malaysia). .. The construction of paired-end sequencing libraries using NEBNext® UltraTM IIDNA Library Prep Kit (Cat No. E7645), with insert sizes of 350 bp, adhered to the standard protocol provided by New England Biolabs (Massachusetts, United States) and Illumina (San Diego, CA, USA). .. The sequencing process was carried out using the Illumina NovaSeq X sequencing platform, with a read length of 150 bp at each end.

Article Title: alpseq: an open-source workflow to turbocharge nanobody discovery with high-throughput sequencing
Article Snippet: Three different pairs of restriction enzymes were used to maintain sequence diversity at both ends, which is critical for Illumina sequencing with the 2-colour chemistry ( ). .. Paired-end sequencing libraries were prepared using the NEBNext Multiplex Oligos for Illumina (Cat # E7395) in a PCR-free manner according to the manufacturer’s instructions. .. The samples were sizeselected using AMPure XP beads with a 0.7x ratio (Cat # A63881) and quantified by qPCR using the KAPA Library Quantification Kit (Cat # KK4873) and sequenced on an Illumina NextSeq 2000 instrument with the P1 2×300 cycle kit (with latest XLEAP-SBS chemistry) and a 20% PhiX spikein.

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Multiplex Assay:

Polymerase Chain Reaction:

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A Mock-infected and WT Salmonella -infected macrophages 5 min post infection. Actin (red); Salmonella (green). Arrows indicate membrane-associated bacteria. B Percentage of macrophages interacting with WT Salmonella during the first 15 min, as determined by immunofluorescence microscopy and stratified by membrane-associated or intracellular bacteria. Three biological replicates. C Salmonella colony forming units (CFU) recovered 75 min post mock, WT and SPI-1 deficient (Δ prgJ ) Salmonella infection per macrophage. As expected, no CFUs were detected in mock infection (n.d.). Significance determined by two-sided Student’s t -test; two biological replicates; three technical triplicates each. D Cytotoxicity of WT or Δ prgJ Salmonella , or mock infection of macrophages as determined by lactate dehydrogenase (LDH) release into culture supernatant. Four technical replicates, representative of 3 independent experiments. B – D Error bars show mean ± SEM. E Experimental outline detailing the preparation of ribosome profiling and RNA-Seq libraries from infected macrophages. Details are provided in the materials and methods (RPF: ribosome-protected RNA fragments, NGS: next-generation sequencing). F Diagram illustrating the relationship between mRNA abundance (RNA-Seq), protein synthesis (Ribo-Seq), and translational efficiency (TE). E , F Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . G Changes in mRNA abundance (left) and TE (right) on infection with WT or Δ prgJ Salmonella compared to mock infection. Genes upregulated transcriptionally in both (log 2 FC > 1) are shown in red. H Normalized relative mRNA abundance, protein synthesis, and TE of transcriptionally upregulated mRNAs from ( G ). Hierarchical clustering was used to order genes by mRNA abundance across all conditions (left). Genes with low read counts (i.e., normalized RNA-Seq and Ribo-Seq counts <5; shown in grey) are too poorly expressed to reliably calculate TE values. I Normalized Ribo-Seq read count and RNA-Seq coverage of Tnf and Zfp36 , which are known to have increased TE on exposure to bacterial PAMPs. Ribo-Seq reads are represented by their P site position, colored by their reading frame relative to the start of the coding sequence, represented by the bar above each plot with the start and stop positions indicated on the x-axis. Rpl4 is presented as a control gene expressed in all samples. P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparisons . All adjusted P < 10⁻ 12 .

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: A Mock-infected and WT Salmonella -infected macrophages 5 min post infection. Actin (red); Salmonella (green). Arrows indicate membrane-associated bacteria. B Percentage of macrophages interacting with WT Salmonella during the first 15 min, as determined by immunofluorescence microscopy and stratified by membrane-associated or intracellular bacteria. Three biological replicates. C Salmonella colony forming units (CFU) recovered 75 min post mock, WT and SPI-1 deficient (Δ prgJ ) Salmonella infection per macrophage. As expected, no CFUs were detected in mock infection (n.d.). Significance determined by two-sided Student’s t -test; two biological replicates; three technical triplicates each. D Cytotoxicity of WT or Δ prgJ Salmonella , or mock infection of macrophages as determined by lactate dehydrogenase (LDH) release into culture supernatant. Four technical replicates, representative of 3 independent experiments. B – D Error bars show mean ± SEM. E Experimental outline detailing the preparation of ribosome profiling and RNA-Seq libraries from infected macrophages. Details are provided in the materials and methods (RPF: ribosome-protected RNA fragments, NGS: next-generation sequencing). F Diagram illustrating the relationship between mRNA abundance (RNA-Seq), protein synthesis (Ribo-Seq), and translational efficiency (TE). E , F Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . G Changes in mRNA abundance (left) and TE (right) on infection with WT or Δ prgJ Salmonella compared to mock infection. Genes upregulated transcriptionally in both (log 2 FC > 1) are shown in red. H Normalized relative mRNA abundance, protein synthesis, and TE of transcriptionally upregulated mRNAs from ( G ). Hierarchical clustering was used to order genes by mRNA abundance across all conditions (left). Genes with low read counts (i.e., normalized RNA-Seq and Ribo-Seq counts <5; shown in grey) are too poorly expressed to reliably calculate TE values. I Normalized Ribo-Seq read count and RNA-Seq coverage of Tnf and Zfp36 , which are known to have increased TE on exposure to bacterial PAMPs. Ribo-Seq reads are represented by their P site position, colored by their reading frame relative to the start of the coding sequence, represented by the bar above each plot with the start and stop positions indicated on the x-axis. Rpl4 is presented as a control gene expressed in all samples. P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparisons . All adjusted P < 10⁻ 12 .

Article Snippet: Preparation of mRNA 3ʹ end sequencing libraries was performed using QuantSeq 3ʹ mRNA-Seq Library Prep Kit (Lexogen) with TRIzol extracted RNA and libraries were sequenced by Novogene Ltd.

Techniques: Infection, Membrane, Bacteria, Immunofluorescence, Microscopy, RNA Sequencing, Next-Generation Sequencing, Sequencing, Control, Two Tailed Test

A Normalized relative mRNA abundance, protein synthesis, and TE of mRNAs with log 2 FC in TE of WT over Δ prgJ infected macrophages greater than 1.5 and at least 50 normalized Ribo-Seq counts in WT infection. Transcripts are ordered by hierarchical clustering of TE across all conditions (left). B Normalized Ribo-Seq read count and RNA-Seq transcript coverage of Egr1 , Il1b and Nr4a1 as in Fig. . Genes with low read counts (i.e., those with normalized RNA-Seq and Ribo-Seq counts <5; shown as grey bars in B ) are too poorly expressed to permit TE values to be reliably calculated. P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparison . All adjusted P < 10⁻ 14 .

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: A Normalized relative mRNA abundance, protein synthesis, and TE of mRNAs with log 2 FC in TE of WT over Δ prgJ infected macrophages greater than 1.5 and at least 50 normalized Ribo-Seq counts in WT infection. Transcripts are ordered by hierarchical clustering of TE across all conditions (left). B Normalized Ribo-Seq read count and RNA-Seq transcript coverage of Egr1 , Il1b and Nr4a1 as in Fig. . Genes with low read counts (i.e., those with normalized RNA-Seq and Ribo-Seq counts <5; shown as grey bars in B ) are too poorly expressed to permit TE values to be reliably calculated. P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparison . All adjusted P < 10⁻ 14 .

Techniques: Infection, RNA Sequencing, Two Tailed Test, Comparison

A mRNA abundance, protein synthesis, and translational efficiency (TE) of Egr1 from Fig. . In conditions with low Egr1 expression TE cannot be reliably calculated (gray bar). P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparison . P = 1.02 × 10⁻ 15 B Egr1 transcript abundance during WT and SPI-1 deficient mutant (Δ prgJ ) infection, normalized to the housekeeping gene Supt16 and relative to their abundance in cells prior to infection; n = 2. C Immunoblot following EGR1 and GAPDH protein abundance across WT and Δ prgJ infection time course. An equal amount of cellular protein was loaded per lane. Samples derive from the same experiment and blots were processed in parallel. Representative of 2 independent experiments. D Quantification of EGR1 abundance from C normalized to GAPDH. E Immunoblot showing EGR1 expression in WT (EGR1 WT ) and EGR1 knockout (EGR1 KO ) macrophages infected with WT Salmonella at 120 min post infection. Representative of 2 independent experiments. F Cytotoxicity of WT or mock infection of EGR1 WT and EGR1 KO macrophages determined by lactate dehydrogenase (LDH) release into culture supernatant; n = 3. G IL-1β concentration in culture supernatant from infected EGR1 KO and EGR1 WT macrophages. 2 biological replicates except for 240 min WT Salmonella infection where there were 3 for EGR1 WT and 4 for EGR1 KO ; significance determined using one-sided Student’s t -test; P = 0.048; error bars show mean ± SEM. H Normalized mRNA abundance of transcripts upregulated (log 2 FC > 2) in EGR1 KO compared to EGR1 WT macrophages at any time point in WT Salmonella infection. Genes are ordered by hierarchical clustering (left). I The 10 most significantly enriched gene ontology biological process terms in genes identified in H. P values were determined with Fisher’s one-tailed test with Benjamini-Hochberg correction for multiple comparisons.

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: A mRNA abundance, protein synthesis, and translational efficiency (TE) of Egr1 from Fig. . In conditions with low Egr1 expression TE cannot be reliably calculated (gray bar). P values were estimated using the two-tailed Bayesian framework from Xtail, comparing mRNA and RPF log₂FC and log 2 ratios across two pipelines and reporting the more conservative result with Benjamini-Hochberg correction for multiple comparison . P = 1.02 × 10⁻ 15 B Egr1 transcript abundance during WT and SPI-1 deficient mutant (Δ prgJ ) infection, normalized to the housekeeping gene Supt16 and relative to their abundance in cells prior to infection; n = 2. C Immunoblot following EGR1 and GAPDH protein abundance across WT and Δ prgJ infection time course. An equal amount of cellular protein was loaded per lane. Samples derive from the same experiment and blots were processed in parallel. Representative of 2 independent experiments. D Quantification of EGR1 abundance from C normalized to GAPDH. E Immunoblot showing EGR1 expression in WT (EGR1 WT ) and EGR1 knockout (EGR1 KO ) macrophages infected with WT Salmonella at 120 min post infection. Representative of 2 independent experiments. F Cytotoxicity of WT or mock infection of EGR1 WT and EGR1 KO macrophages determined by lactate dehydrogenase (LDH) release into culture supernatant; n = 3. G IL-1β concentration in culture supernatant from infected EGR1 KO and EGR1 WT macrophages. 2 biological replicates except for 240 min WT Salmonella infection where there were 3 for EGR1 WT and 4 for EGR1 KO ; significance determined using one-sided Student’s t -test; P = 0.048; error bars show mean ± SEM. H Normalized mRNA abundance of transcripts upregulated (log 2 FC > 2) in EGR1 KO compared to EGR1 WT macrophages at any time point in WT Salmonella infection. Genes are ordered by hierarchical clustering (left). I The 10 most significantly enriched gene ontology biological process terms in genes identified in H. P values were determined with Fisher’s one-tailed test with Benjamini-Hochberg correction for multiple comparisons.

Techniques: Expressing, Two Tailed Test, Comparison, Mutagenesis, Infection, Western Blot, Quantitative Proteomics, Knock-Out, Concentration Assay, One-tailed Test

Data from two biological replicates. A Outline of infection time course. Primary bone marrow-derived macrophages were generated from mice and infected with WT or Δ prgJ mutant Salmonella . Gentamicin was used to kill extracellular bacteria at 15 min, and its concentration was reduced after 1 h. Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . B Cytotoxicity of WT Salmonella or mock infection of primary macrophages determined by lactate dehydrogenase (LDH) release into culture supernatant; 2 mice, technical duplicates. C Transient expression dynamics of select WT Salmonella genes at different Salmonella culture optical densities (OD) (Supplementary Data ) and during the 4 h macrophage infection, at the level of mRNA abundance, protein synthesis, and translation efficiency.

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: Data from two biological replicates. A Outline of infection time course. Primary bone marrow-derived macrophages were generated from mice and infected with WT or Δ prgJ mutant Salmonella . Gentamicin was used to kill extracellular bacteria at 15 min, and its concentration was reduced after 1 h. Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . B Cytotoxicity of WT Salmonella or mock infection of primary macrophages determined by lactate dehydrogenase (LDH) release into culture supernatant; 2 mice, technical duplicates. C Transient expression dynamics of select WT Salmonella genes at different Salmonella culture optical densities (OD) (Supplementary Data ) and during the 4 h macrophage infection, at the level of mRNA abundance, protein synthesis, and translation efficiency.

Techniques: Infection, Derivative Assay, Generated, Mutagenesis, Bacteria, Concentration Assay, Expressing

Data from two biological replicates. A Differential regulation of host gene expression represented as the log 2 FC of translation efficiency (TE) versus log 2 FC mRNA abundance in WT Salmonella infection compared to Δ prgJ infection. Dashed orange lines show the log 2 FC cutoffs (±2) used to select differentially expressed genes, and genes that pass these thresholds are colored. Only genes with log 2 FC in mRNA and TE between −5 and 5 are shown here (see Fig for uncropped plots). Icons created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . Number of genes differentially expressed between WT and Δ prgJ Salmonella infection at both the TE ( B ) and mRNA ( C ) levels. D Top 10 enriched GO molecular function terms in differentially expressed genes at both the TE (right) and mRNA (left) levels. Genes were grouped by the timing of differential expression: at or before 60 min, and after 60 min post infection. P values were determined with Fisher’s one-tailed test with Benjamini-Hochberg correction for multiple comparisons.

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: Data from two biological replicates. A Differential regulation of host gene expression represented as the log 2 FC of translation efficiency (TE) versus log 2 FC mRNA abundance in WT Salmonella infection compared to Δ prgJ infection. Dashed orange lines show the log 2 FC cutoffs (±2) used to select differentially expressed genes, and genes that pass these thresholds are colored. Only genes with log 2 FC in mRNA and TE between −5 and 5 are shown here (see Fig for uncropped plots). Icons created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . Number of genes differentially expressed between WT and Δ prgJ Salmonella infection at both the TE ( B ) and mRNA ( C ) levels. D Top 10 enriched GO molecular function terms in differentially expressed genes at both the TE (right) and mRNA (left) levels. Genes were grouped by the timing of differential expression: at or before 60 min, and after 60 min post infection. P values were determined with Fisher’s one-tailed test with Benjamini-Hochberg correction for multiple comparisons.

Techniques: Gene Expression, Infection, Quantitative Proteomics, One-tailed Test

A Experimental outline illustrating the effect of the SptP-GFP injectisome blocking substrate on protein export via the injectisome. Salmonella cells not producing the blocking substrate can transport effector proteins into host cells (unobstructed injectisome, left). Salmonella cells expressing the injectisome blocking substrate before infection are unable to transport effector subunits via the injectisome (pre-blocked injectisome, middle). Salmonella cells were also incubated with macrophages for 5 min before inducing expression of the injectisome blocking substrate such that injectisomes can engage with macrophage cells but secretion of effectors proteins from Salmonella into macrophages is blocked at 5 min (injectisome blocked at 5 min, right). Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . B Secretion analysis of WT Salmonella either expressing the SptP-GFP injectisome blocking subunit (+) or carrying the empty vector (-). Whole cell (cell) and secreted proteins (sec) from late-log-phase cultures were separated by SDS-PAGE and immunoblotted with anti-Myc-tag (SptP-GFP), anti-SipC, or anti-GroEL antisera. Representative of 3 independent experiments. C Cytotoxicity following infection with and without injectisome blockage as described in A. LDH concentration was determined in cell culture supernatants at 120 min post infection, LDH at 0 min was subtracted and values shown are relative to total cell lysis. Significance determined by one-sided Student’s t -test with Benjamini-Hochberg adjusted P values of: Mock: Unobstructed = 1.0 × 10⁻ 3 , Mock: Pre-blocked = 9.4 × 10⁻ 1 , Mock: Blocked at 5 min = 1.7 × 10⁻ 1 , and Unobstructed: Blocked at 5 min = 9.5 × 10 − 4 ; n = 3 biological replicates; data shows mean ± SEM. D Egr1 transcript abundance across a Salmonella infection time course with blockage of the SPI-1 injectisome induced as indicated. E A capped, polyadenylated mRNA reporter (top) encoding firefly luciferase flanked by the Egr1 5ʹ and 3ʹ UTRs was transfected into cells, which was followed by Salmonella infection with blockage of the SPI-1 injectisome induced as indicated (MOI 10). Luciferase activity was assayed at 2 h, normalized by relative total protein and reporter mRNA abundance to determine the rate of reporter mRNA translation (bottom). Error bars show standard error of quotients; significance determined by two-sided Student’s t -test with Benjamini-Hochberg adjusted P values of: Mock: Pre-blocked = 9.7 × 10⁻ 2 and Mock: Blocked at 5 min = 3.0 × 10⁻ 2 ; n = 5 biological replicates; error bars show mean ± SEM.

Journal: Nature Communications

Article Title: The Salmonella pathogenicity island 1 injectisome reprograms host cell translation to evade the inflammatory response

doi: 10.1038/s41467-025-64744-w

Figure Lengend Snippet: A Experimental outline illustrating the effect of the SptP-GFP injectisome blocking substrate on protein export via the injectisome. Salmonella cells not producing the blocking substrate can transport effector proteins into host cells (unobstructed injectisome, left). Salmonella cells expressing the injectisome blocking substrate before infection are unable to transport effector subunits via the injectisome (pre-blocked injectisome, middle). Salmonella cells were also incubated with macrophages for 5 min before inducing expression of the injectisome blocking substrate such that injectisomes can engage with macrophage cells but secretion of effectors proteins from Salmonella into macrophages is blocked at 5 min (injectisome blocked at 5 min, right). Created in BioRender. Chung, B. (2025) https://BioRender.com/sjej7aq . B Secretion analysis of WT Salmonella either expressing the SptP-GFP injectisome blocking subunit (+) or carrying the empty vector (-). Whole cell (cell) and secreted proteins (sec) from late-log-phase cultures were separated by SDS-PAGE and immunoblotted with anti-Myc-tag (SptP-GFP), anti-SipC, or anti-GroEL antisera. Representative of 3 independent experiments. C Cytotoxicity following infection with and without injectisome blockage as described in A. LDH concentration was determined in cell culture supernatants at 120 min post infection, LDH at 0 min was subtracted and values shown are relative to total cell lysis. Significance determined by one-sided Student’s t -test with Benjamini-Hochberg adjusted P values of: Mock: Unobstructed = 1.0 × 10⁻ 3 , Mock: Pre-blocked = 9.4 × 10⁻ 1 , Mock: Blocked at 5 min = 1.7 × 10⁻ 1 , and Unobstructed: Blocked at 5 min = 9.5 × 10 − 4 ; n = 3 biological replicates; data shows mean ± SEM. D Egr1 transcript abundance across a Salmonella infection time course with blockage of the SPI-1 injectisome induced as indicated. E A capped, polyadenylated mRNA reporter (top) encoding firefly luciferase flanked by the Egr1 5ʹ and 3ʹ UTRs was transfected into cells, which was followed by Salmonella infection with blockage of the SPI-1 injectisome induced as indicated (MOI 10). Luciferase activity was assayed at 2 h, normalized by relative total protein and reporter mRNA abundance to determine the rate of reporter mRNA translation (bottom). Error bars show standard error of quotients; significance determined by two-sided Student’s t -test with Benjamini-Hochberg adjusted P values of: Mock: Pre-blocked = 9.7 × 10⁻ 2 and Mock: Blocked at 5 min = 3.0 × 10⁻ 2 ; n = 5 biological replicates; error bars show mean ± SEM.

Techniques: Blocking Assay, Expressing, Infection, Incubation, Plasmid Preparation, SDS Page, Concentration Assay, Cell Culture, Lysis, Luciferase, Transfection, Activity Assay

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