preimmune mouse serum  (Millipore)

Journal: Nature Communications

Article Title: Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria

doi: 10.1038/s41467-024-44774-6

Figure Lengend Snippet: A Spot colony morphology of the wide type and its ydeH -overexpressed strains, hadD Msm knock-out and its ydeH -overexpression strains. B Biofilm formation of the WT, ydeH , hadD Msm KO, and hadD Msm KO/ ydeH strains. C Quantitation of biofilm biomass by crystal violet staining of the WT, ydeH , hadD Msm KO, and hadD Msm KO/ ydeH strains ( n = 3, biological replicates). Two-tailed t-tests were performed for statistical analysis (*** p = 0.0004). D RT-PCR quantitation of hadD Msm transcription in the ydeH , ydeH (mut)-overexpressed M. smegmatis strains. Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). RT-PCR and gel analysis were performed under the same conditions and each experiment was performed three independent biological replicates. no RT was genomic DNA contamination control. E ChIP assays for the effect of c-di-GMP on the intracellular hadD Msm p-binding activity of Lsr2 Msm in M. smegmatis . The input (5%) indicated that the supernatant of disrupted cells was diluted to 5%, ChIP using preimmune (P) or immune (I) sera raised against HisLsr2 Msm . DNA of the input (5%), P, and I were used as temples for PCR (the right panel) ( n = 3, biological replicates) and RT-qPCR (the light panel). M: DNA Marker. Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). F β-galactosidase activity assays. The effect of c-di-GMP on Lsr2 Msm regulates the expression of hadD Msm was assayed by overexpressing pMV261- hadD Msm p- ydeH - lacZ and pMV261- hadD Msm p- ydeH (mut)- lacZ plasmids in the Msm/WT, lsr2 Msm KO strains ( n = 3, biological replicates). None promoter- lacZ and hsp60 p- lacZ plasmids overexpression were used as controls. Two-tailed Student’s t-tests were performed for statistical analysis (* p = 0.0414, **** p < 0.0001). Data were presented as mean ± SD of ( C ), ( D ), ( E ), ( F ). The source data were provided in the Source data file.

Article Snippet: The 900 µL supernatant was incubated with 1:2,000 dilution of mouse 6*His antibodies (#CSB-MA000011M0m, CUSABIO) or preimmune mouse serum (#NS03L, Sigma-Aldrich) for 3 h at 4 °C.

Techniques: Knock-Out, Over Expression, Quantitation Assay, Staining, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay, Quantitative RT-PCR, Marker, Expressing

Journal: Nature Communications

Article Title: Lsr2 acts as a cyclic di-GMP receptor that promotes keto-mycolic acid synthesis and biofilm formation in mycobacteria

doi: 10.1038/s41467-024-44774-6

Figure Lengend Snippet: A ITC assays for the interaction between Lsr2 Mtb and c-di-GMP. Original titration data and integrated heat measurements are shown in the upper and lower plots. B Spot colony morphology of the wide type, hadD Msm knock-out and hadD Mtb complementary strains. C Biofilm formation of the WT, hadD Msm KO, and hadD Mtb complementary strains. D Quantitation of biofilm biomass of the WT, hadD Msm KO, and hadD Mtb complementary strains by crystal violet staining ( n = 3, biological replicates). Two-tailed t-tests were performed for statistical analysis (**** p < 0.0001, *** p = 0.0004). E EMSA assays for the effect of c-di-GMP on hadD BCG promoter DNA-binding activity of Lsr2 BCG . hadD BCG p was co-incubated with increasing concentration of Lsr2 BCG (lanes 2–5). Three independent experiments were performed. F RT-PCR for transcriptional analysis of hadD BCG in the WT, lsr2 BCG KO M. bovis BCG strains ( n = 3, biological replicates). Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). RT-PCR and gel analysis were performed under the same conditions. G Spot colony morphology of the WT and lsr2 BCG knock-out BCG strains on 7H10 plates. H RT-PCR for transcriptional analysis of hadD BCG in the ydeH (mut), ydeH -overexpressed M. bovis BCG strains ( n = 3, biological replicates). Two-tailed Student’s t-tests were performed for statistical analysis (**** p < 0.0001). I ChIP assays for the effect of c-di-GMP on the intracellular DNA-binding activity of Lsr2 BCG in the M. bovis BCG strains. The input (5%) indicated that the supernatant of disrupted cells was diluted to 5%, ChIP using preimmune (P) or immune (I) sera raised against HisLsr2 BCG . DNA sample of the input (5%), P, and I were used as temples for PCR (the right panel) ( n = 3, biological replicates) and were quantified using RT-qPCR (the light panel). M: DNA marker. Two-tailed Student’s t-tests were performed to for statistical analysis (**** p < 0.0001). Data of figures ( D ), ( F ), ( H ), and ( I ) were presented as mean ± SD. The source data were provided in the Source data file.

Article Snippet: The 900 µL supernatant was incubated with 1:2,000 dilution of mouse 6*His antibodies (#CSB-MA000011M0m, CUSABIO) or preimmune mouse serum (#NS03L, Sigma-Aldrich) for 3 h at 4 °C.

Techniques: Titration, Knock-Out, Quantitation Assay, Staining, Two Tailed Test, Binding Assay, Activity Assay, Incubation, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Marker

Journal: Journal of Virology

Article Title: Host IQGAP1 and Ebola Virus VP40 Interactions Facilitate Virus-Like Particle Egress

doi: 10.1128/JVI.00470-13

Figure Lengend Snippet: EBOV VP40 interacts with endogenous IQGAP1. Cell proteins from mock-transfected cells (pCAGGS vector alone; lanes 3 and 4) or pCAGGS-VP40-transfected cells (lanes 1 and 2) were first immunoprecipitated (IP) with either mouse preimmune (control; lanes 2 and 4) serum (Invitrogen) or monoclonal anti-IQGAP1 (lanes 1 and 3) antiserum (Invitrogen) as indicated, and EBOV VP40 was detected in the precipitated samples by Western blot (WB) analysis using anti-VP40 antiserum (top panel). Controls for expression of endogenous IQGAP1, β-actin, and VP40 are shown. Ab, antibody.

Article Snippet: Cell proteins from mock-transfected cells (pCAGGS vector alone; lanes 3 and 4) or pCAGGS-VP40-transfected cells (lanes 1 and 2) were first immunoprecipitated (IP) with either mouse preimmune (control; lanes 2 and 4) serum (Invitrogen) or monoclonal anti-IQGAP1 (lanes 1 and 3) antiserum (Invitrogen) as indicated, and EBOV VP40 was detected in the precipitated samples by Western blot (WB) analysis using anti-VP40 antiserum (top panel).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Host IQGAP1 and Ebola Virus VP40 Interactions Facilitate Virus-Like Particle Egress

doi: 10.1128/JVI.00470-13

Figure Lengend Snippet: EBOV VP40 interacts with endogenous IQGAP1 in Tsg101-suppressed cells. Cells were cotransfected with EBOV VP40 and Tsg101-specific siRNAs (Santa Cruz) (lanes 1 and 2) or random siRNAs (Santa Cruz) (lanes 3 and 4). Transfected cell extracts were first immunoprecipitated with either mouse preimmune (control; lanes 2 and 4) serum (Invitrogen) or monoclonal anti-IQGAP1 (lanes 1 and 3) antiserum (Invitrogen) as indicated, and EBOV VP40 was detected in the precipitated samples by Western analysis using polyclonal anti-VP40 antiserum (top panel). Controls for expression of endogenous IQGAP1, β-actin, VP40, and Tsg101 are shown.

Article Snippet: Cell proteins from mock-transfected cells (pCAGGS vector alone; lanes 3 and 4) or pCAGGS-VP40-transfected cells (lanes 1 and 2) were first immunoprecipitated (IP) with either mouse preimmune (control; lanes 2 and 4) serum (Invitrogen) or monoclonal anti-IQGAP1 (lanes 1 and 3) antiserum (Invitrogen) as indicated, and EBOV VP40 was detected in the precipitated samples by Western blot (WB) analysis using anti-VP40 antiserum (top panel).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: SMAD3 Deficiency Promotes Inflammatory Aortic Aneurysms in Angiotensin II–Infused Mice Via Activation of iNOS

doi: 10.1161/JAHA.113.000269

Figure Lengend Snippet: Molecular mechanism by which SMAD3 deficiency causes enhanced iNOS‐derived NO production in aorta. A and B, Fluorescence staining for NO, iNOS, and F4/80 in vehicle‐ (A) and AngII‐infused (B) WT and S3KO aortas. Dotted lines demarcate adventitial layer from medial layer. Scale bars=50 μm. C, Western blots showing increased C/EBPβ, phosphorylated NF‐κB, and iNOS in S3KO aorta. Beta‐tubulin served as a loading control. D, In vivo ChIP of C/EBP binding site within the mouse iNOS promoter and a control region 2 kb upstream of the C/EBP site using antibodies against C/EBPβ or preimmune IgG. The gene fragments in the immunoprecipitated chromatin were quantified by RT‐qPCR. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). E, Re‐ChIP was performed using antibodies against coactivator p300. AngII indicates angiotensin II; iNOS indicates inducible nitric oxide synthase; WT, wild type; NO, nitric oxide; S3KO, SMAD3 knockout; C/EBP, CCAAT/enhancer binding protein; L, lumen; m, tunica media; a, tunica adventitia; NF‐κB, nuclear factor‐kappaB.

Article Snippet: Antibodies against CCAAT/enhancer binding protein (C/EBP)–β (#sc‐150), p300 (#sc‐585), α‐smooth muscle actin (α‐SMA [#sc‐69972]), β‐tubulin (#sc‐9104), horseradish peroxidase (HRP)–conjugated goat anti‐mouse IgG (#sc‐2005), and preimmune serum (#sc‐2338) were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Fluorescence, Staining, Western Blot, Control, In Vivo, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Knock-Out