Journal: Life Science Alliance

Article Title: Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

doi: 10.26508/lsa.202302164

Figure Lengend Snippet: (A) HEK 293T cells were transfected by constructs coding for ECFP or ECFP-tagged peptides (sequences listed in the table), or SLAMF1 or TRAM FLAG proteins. After 48 h, cell lysates were prepared, and lysates containing ECFP- or ECFP-tagged peptides were normalized for the comparable expression levels, which were controlled by anti GFP WB (top panel). Whole-cell lysates from TRAM FLAG or SLAMF1 overexpressing cells were also analyzed by WB. Lysates with ECFP and ECFP peptides were mixed with equal amounts of lysates with overexpressed TRAM FLAG and SLAMF1, and incubated with anti-FLAG beads for 2 h, followed by WB analysis of co-precipitated proteins. Samples shown for anti-GFP, anti-SLAMF1, and anti-TRAM WBs were loaded and transferred to the same membranes, with several bands excised on the presented images. Quantification of SLAMF1 protein to TRAM FLAG ratio in IPs is shown on graph. One of three independent experiments. (B) Anti-FLAG IPs for WT or P333T mutant SLAMF1 FLAG with YFP-tagged TRAM protein. Input (Whole-cell lysates) comprised of 7.5% from the sample used for IP. Correlation of GFP signal intensity to FLAG signal intensity is shown on the graph. Data presented as mean ± SD for three independent experiments, significance evaluated by t test with Welch’s correction (* P < 0.05). (C) Quantification by qRT-PCR of IFNβ mRNA expression in THP-1 cells pretreated for 30 min with variable concentrations of control Arg11 or P7-Arg11 peptides (5, 10 or 20 μM), followed by stimulation with LPS (100 ng/ml). Results presented as mean ± SD for three biological replicates (one of three experiments). (C, D) Western blot analysis of p-STAT1 expression in THP-1 cells pretreated with peptides and stimulated with LPS as in (C). β-tubulin WB used for loading control. (E) Quantification of IFNβ and TNF mRNA expression by qRT-PCR in THP-1 cells pretreated by peptide solvents (water, H 2 O or DMSO) or peptides (15 μM) for 30 min and stimulated with LPS for indicated time points. Data for each time point are normalized to water (H 2 O) and presented as ratio between mRNA expression values of cells treated with peptide to values for cells treated with water. Data presented as mean relative fold change + SD (data from 6–11 independent experiments). (E, F) Cell death evaluated by LDH release assay in supernatants of cells used for qRT-PCR analysis in (E). Data presented as mean for percentage of dead cells + SD. (E, F) Statistical testing was done by mixed effect model on log-transformed data (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Source data are available for this figure.

Article Snippet: IPs were performed using 35 μl/IP of anti-FLAG M2 affinity agarose solution (Sigma-Aldrich, Merck), beads washed by lysis buffer before adding respective lysates.

Techniques: Transfection, Construct, Expressing, Incubation, Mutagenesis, Quantitative RT-PCR, Western Blot, Lactate Dehydrogenase Assay, Transformation Assay

Journal: Life Science Alliance

Article Title: Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

doi: 10.26508/lsa.202302164

Figure Lengend Snippet: (A, B, C, D) HEK 293T cells were co-transfected by SLAMF1 and TRAM FLAG (A), TRAM YFP and TLR4 FLAG (B), TRIF HA and TRAM FLAG (C) or FIP2 EGFP and TRAM FLAG (D) for 48 h, followed by treatment of cells by 30 μM peptides C3-Pen (control, C3) or P7-Pen (P7) for 1 h, lysis of cells and anti-FLAG IPs for 3 h. Whole-cell lysates loaded for the input control, where input represents 7.5% from the total sample used for IP. (A, B, C, D) Ratio between co-precipitated proteins to FLAG-tagged proteins was quantified for three to four independent experiments and presented on graphs to the right from the respective WB (A, B, C, D). Significance evaluated by t test with Welch’s correction, significance levels (** P < 0.01, **** P < 0.0001, ns, nonsignificant). Source data are available for this figure.

Article Snippet: IPs were performed using 35 μl/IP of anti-FLAG M2 affinity agarose solution (Sigma-Aldrich, Merck), beads washed by lysis buffer before adding respective lysates.

Techniques: Transfection, Lysis

Journal: Life Science Alliance

Article Title: Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

doi: 10.26508/lsa.202302164

Figure Lengend Snippet: (A) HEK-Blue IL-1R cells were stimulated by recombinant human IL-1β for indicated time points, followed by Western blot analysis of p38 MAPK phosphorylation and IRAK1 posttranslational modifications. Short and long exposure blots shown for IRAK1 for better visualization of 80 kD and 100 kD IRAK1 forms that correspond to unmodified or phosphorylated/ubiquitinated IRAK1. β-tubulin WB used for loading control. (B) Western blot analysis of TRL2- and TLR4-mediated TBK1 and p38 MAPK phosphorylation in human monocytes. Cells were pretreated with 15 μM peptides for 30 min, followed by stimulation with TLR2 ligand FSL-1 (100 ng/ml) or TLR4 ligand LPS (100 ng/ml), and unstimulated samples analyzed for negative control. PCNA WB served for loading control. (C, D) Anti-FLAG IPs from HEK 293T cells, transfected as indicated by constructs coding for human proteins TLR4 FLAG (C) or TLR2 FLAG (D), MyD88 CFP and TIRAP HA (C, D) Whole-cell lysates loaded for input control, with input (whole-cell lysates) to IP loading ratio 1:10.

Article Snippet: IPs were performed using 35 μl/IP of anti-FLAG M2 affinity agarose solution (Sigma-Aldrich, Merck), beads washed by lysis buffer before adding respective lysates.

Techniques: Recombinant, Western Blot, Negative Control, Transfection, Construct

Journal: Life Science Alliance

Article Title: Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

doi: 10.26508/lsa.202302164

Figure Lengend Snippet: (A, B) Endogenous TIRAP (A) or anti -FLAG (B) IPs from lysates of THP-1 TLR4 FLAG cells. (C) Endogenous anti-TIRAP IPs from lysates of primary human monocytes. (A, B, C) Cells were pretreated by 15 μM peptides C3-Pen (C3) or P7-Pen (P7) and stimulated by LPS (100 ng/ml) for indicated time, with unstimulated cells used for negative control. (A, B, C) Cellular lysates were loaded for input control, with WBs performed for MyD88, TIRAP (A, B, C), FLAG (B), and IRAK1 (C). Input (whole-cell lysates) represents 4.6% from the total sample used for IP. (A, B, C) Representative experiments from a total of three for each experimental setting (A, B, C). (D, E) Western blot analysis of lysates and anti-FLAG IPs from HEK 293T cells, overexpressing TIRAP FLAG and MyD88 CFP (D) or TLR4 FLAG and TIRAP HA (E), performed in 48 h after transfection and after 1 h of pretreatment of cells by 30 μM peptides. Whole-cell lysates loaded for input control, which represents 14.5% from the total sample used for IP. Ratio between co-precipitated proteins to FLAG-tagged proteins was quantified for three to four independent experiments and presented on graphs to the right from the respective WB. Statistical significance calculated using t test with Welch’s correction, significance levels: *** P < 0.001, ns, nonsignificant. Source data are available for this figure.

Article Snippet: IPs were performed using 35 μl/IP of anti-FLAG M2 affinity agarose solution (Sigma-Aldrich, Merck), beads washed by lysis buffer before adding respective lysates.

Techniques: Negative Control, Western Blot, Transfection

Journal: Life Science Alliance

Article Title: Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo

doi: 10.26508/lsa.202302164

Figure Lengend Snippet: (A) HEK 293T cells were transfected by plasmids coding for human or murine TRAM FLAG proteins for 48 h, followed by lysing of cells and PDs by biotinylated peptides Pen or P7-Pen (P7) fixed on NeutrAvidin beads for 1 h. Co-precipitation of FLAG-tagged TRAM orthologs with peptides was addressed by anti-FLAG WB, with WCLs for input control, which represents 14.5% from the total sample used for PD. Sequence alignments for human and murine orthologs of SLAMF1 (in the domain used for peptide design) and TRAM (domain involved in interaction with SLAMF1) are shown below the WB panels. (B) Murine immortalized bone marrow-derived macrophages B6 WT were pretreated with 10 μM peptides or a similar amount of sterile water (solvent, H 2 O) for 30 min and stimulated by LPS (100 ng/ml) for the indicated time. Lysates were analyzed by WB to address TLR4-mediated phosphorylation of TAK1, IκBα, p38 MAPK, TBK1 or posttranslational modifications of IRAK1 (followed by disappearance of the 80-kD band), and β-tubulin WB was used for loading control. (B, C) Quantification of Ifnβ , Tnf , and Il-1β mRNA expression by qRT-PCR in B6 WT cells pretreated by peptides or solvent and stimulated by LPS as in (B). Data presented as fold change when compared with unstimulated sample pretreated with water, mean relative fold change ± SD (n = 3). Statistical testing was done by two-way RM-ANOVA. (D) C57BL/6J mice were injected i.p. with repurified Sigma smooth E.coli 0111:B4 LPS (20 μg/g) or PBS. Peptides (2.5 nmol/g of animal weight, 8.34 or 8.5 mg/kg for C3-Pen and P7-Pen, respectively) were injected i.p. 1 h before injection of LPS. (D, E) Probability of survival is shown on (D), and body temperature measurements are shown on (E). Each “x” indicates a succumbed mouse at the time point. (D, E) Statistical significance evaluated by log-rank (Mantel–Cox) test with Gehan–Breslow–Wilxocon test (D) or two-way ANOVA for (E). For all graphs, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure.

Article Snippet: IPs were performed using 35 μl/IP of anti-FLAG M2 affinity agarose solution (Sigma-Aldrich, Merck), beads washed by lysis buffer before adding respective lysates.

Techniques: Transfection, Sequencing, Derivative Assay, Sterility, Solvent, Expressing, Quantitative RT-PCR, Injection