rabbit polyclonal antibodies  (Cell Signaling Technology Inc)

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Induced pluripotent stem cell-derived platelets kill multidrug-resistant Staphylococcus aureus via Toll-like receptor 2-MyD88 signaling and immunoglobulin G/FcγRIIA engagement

doi: 10.1016/j.rpth.2026.103374

Figure Lengend Snippet: MyD88 knockout (KO)-induced pluripotent stem cell (iPSC)-derived platelets (iPSC-PLTs) exhibit attenuated responses to multidrug-resistant Staphylococcus aureus (MRSA). (A) Schematic representation of MyD88-KO generation in immortalized megakaryocyte progenitor cell line (imMKCL; Sev2) cells using the clustered regularly interspaced short palindromic repeats/CRISPR-associated prtotein 9 (CRISPR/Cas9) system through a re-reprogramming approach. (B) Western blots confirmed that single guide RNA (sgRNA) 1 targeting exon 1 yielded clone 4 and failed in the KO, but sgRNA2 targeting exon 3 yielded clones 28, 30, and 37, which succeeded in the KO. (C) MRSA-killing assay results for wild-type (WT) and MyD88-KO iPSC-PLTs in the presence of human plasma. (D) Cumulative flow cytometry data of CD62P expression, PAC-1 binding, and annexin V binding in WT and MyD88-KO iPSC-PLTs in the presence of human plasma. Compiled data of 3 independent experiments. Data are presented as means ± SEM. ∗ P < .05, ∗∗ P < .01. DOX, doxycycline; iPLAT, first-in-human clinical trial of iPSC-PLTs; ns, not significant; MK, megakaryocyte.

Article Snippet: The following antibodies were used: anti–β-actin mouse monoclonal antibody (Sigma-Aldrich, #A1978) and anti-MyD88 rabbit polyclonal antibody (Cell Signaling Technology, #3699).

Techniques: Knock-Out, Derivative Assay, CRISPR, Western Blot, Clone Assay, Clinical Proteomics, Flow Cytometry, Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Cilastatin Attenuates Acute Kidney Injury and Reduces Mortality in a Rat Model of Sepsis

doi: 10.3390/ijms26167927

Figure Lengend Snippet: Effects of cilastatin on cecal ligation and puncture (CLP)-induced Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 88 (MyD88) pathway activation. ( A ) Western blot of TLR4 in the renal cortex and ( B ) respective quantification corrected by the loading control. Results are expressed as mean ± SEM, n = 4–5 animals per group, * p ≤ 0.0094 vs. all groups. ( C ) Renal mRNA expression of TLR4 . Results are expressed as mean ± SEM, n = 4–5 animals per group, * p ≤ 0.046 vs. all groups. ( D ) Immunolocalization of TLR4 through immunofluorescence staining (green) in kidney sections (magnification 20× bar 50 µm). Cilastatin significantly decreased the TLR4 levels that were increased by CLP (arrowheads). ( E ) Serum measurement of LPS Binding Protein (LBP). Results are expressed as mean ± SEM, n = 4 animals per group, * p ≤ 0.0074 vs. all groups. ( F ) Western blot of MyD88 in renal cortex and ( G ) densitometric analysis corrected by the loading control. Results are expressed as mean ± SEM, n = 4–5 animals per group, * p ≤ 0.0094 vs. all groups. ( H ) Renal mRNA expression of MyD88 . Results are expressed as mean ± SEM, n = 5–7 animals per group. ( I ) Immunolocalization (upper image) and immunofluorescence staining (lower image) of MyD88 in renal tissue (magnification 20× bar 50 µm) and ( J ) semi-quantification. Results are expressed as mean ± SEM, n = 6 animals per group, * p ≤ 0.0017 vs. all groups. Note increased the staining in CLP rats (arrowhead and asterisks) and the change in the pattern of that staining compared with CLP + cilastatin and control groups. Cilastatin significantly decreased the MyD88 levels that were increased by CLP, although no significant differences in RNA levels were found. The data were analyzed using ANOVA. Cil: cilastatin; a.u., arbitrary units.

Article Snippet: Sections were then incubated overnight at 4 °C in a humidified chamber with primary antibodies diluted in PBS-T, 4% bovine serum albumin, and 1% host serum in which the secondary antibody was obtained as follows: mouse anti-RelA/NF-κB p65 monoclonal antibody (Novus Biologicals, Minneapolis, MN, USA [dilution 1:100], ref. NB100-56712); mouse anti-TLR4 (25) monoclonal antibody (Santa Cruz Biotechnology [dilution 1:50], ref. sc-293072); and rabbit anti-MyD88 polyclonal antibody (Novus Biologicals [dilution 1:50], ref. NB100-56698).

Techniques: Ligation, Activation Assay, Western Blot, Control, Expressing, Immunofluorescence, Staining, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Cilastatin Attenuates Acute Kidney Injury and Reduces Mortality in a Rat Model of Sepsis

doi: 10.3390/ijms26167927

Figure Lengend Snippet: Summary image of the effects of cilastatin on the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 88 (MyD88)/Nuclear Factor-κB/NF-κB) pathway and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome in cecal ligation and puncture (CLP)-induced inflammation and acute kidney injury. The TLR4 signaling complex is organized and localized in the cholesterol lipid rafts on the brush border apical side of renal proximal tubular epithelial cells, geographically close to the renal dehydrodipeptidase I (DHP-I) enzyme. In the left panel, with cholesterol rafts intact, damage-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPS), such as bacterial lipopolysaccharide (LPS), activate the TLR4/MyD 88 pathway, leading to the activation of NF-κB and pro-inflammatory cytokine and chemokine production. NF-κB is also involved in the activation of the NLRP3 inflammasome that leads to the activation of caspase-1, which, in turn, regulates the processing of inflammatory pro-cytokines to active cytokines (such as interleukin (IL)-1β), amplifying inflammatory damage and cell death, which exacerbates kidney injury. In the right panel, cilastatin binding to the membrane DHP-I in cholesterol lipid rafts causes modifications in the rafts, preventing the correct assembly and activation of the TLR4 complex and reducing the activation of NF-κB and the NLRP3 inflammasome. Thus, the renal cell is protected. K+, potassium; Ca2+, calcium; TIRAP, TIR Domain Containing Adaptor, Protein; IκB, inhibitor of nuclear factor kappa B; TNFα, tumor necrosis factor alpha; GSDMD, Gasdermin D.

Article Snippet: Sections were then incubated overnight at 4 °C in a humidified chamber with primary antibodies diluted in PBS-T, 4% bovine serum albumin, and 1% host serum in which the secondary antibody was obtained as follows: mouse anti-RelA/NF-κB p65 monoclonal antibody (Novus Biologicals, Minneapolis, MN, USA [dilution 1:100], ref. NB100-56712); mouse anti-TLR4 (25) monoclonal antibody (Santa Cruz Biotechnology [dilution 1:50], ref. sc-293072); and rabbit anti-MyD88 polyclonal antibody (Novus Biologicals [dilution 1:50], ref. NB100-56698).

Techniques: Binding Assay, Ligation, Activation Assay, Membrane

Journal: Veterinary Sciences

Article Title: Danggui Buxue Decoction Alleviates Inflammation and Oxidative Stress in Mice with Escherichia coli -Induced Mastitis

doi: 10.3390/vetsci12030227

Figure Lengend Snippet: List of primers.

Article Snippet: A rabbit anti-MyD88 antibody (bs-1047R) (1:3000), anti-IKKβ antibody (bs-4880R) (1:6000), and anti-NF-κB p65 antibody (bs-0465R) (1:1000) were purchased from Bioss (Beijing, China).

Techniques: