Journal: bioRxiv

Article Title: A druggable redox switch on SHP1 controls macrophage inflammation

doi: 10.64898/2026.02.19.706790

Figure Lengend Snippet: a, LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation over a time course of 2 h in iBMDMs pre-treated with DMSO or SCA1 (50 μM) for 3 h. Immunoblots shown are representative of three independent experiments. b, LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation in iBMDMs following 3 h pre-incubation with DMSO, SCA1 or its non-covalent reversible analog of SCA1 (SCA1-NC) at the indicated concentrations. Immunoblots shown are representative of three independent experiments. c , SHP1 binding by SCA derivatives in iBMDMs treated at 5 μM followed by competition for SHP1 binding with biotinylated SCA1 (10 μM). d, Stoichiometric engagement score (R) of each cysteine quantified in iBMDM cysteine proteome treated with SCA9 (50 μM), showing SHP1 Cys102 as top hit exhibiting >50% engagement by SCA9. R = (S/N of DMSO) / (S/N of SCA9), where S/N is signal-to-noise ratio. e , LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation in iBMDMs following 3 h pre-incubation with DMSO or SCA derivatives at the indicated concentrations. Immunoblots shown are representative of two independent experiments. f , Pro-inflammatory cytokine IL-6 and TNF levels in cell supernatants of iBMDMs pre-incubated 3 h with SCA1, SCA1-NC or the respective derivatives over a dose response (0.313-80 μM) followed by 6 h LPS stimulation (100 ng/ml). Normalized percent cytokine production as a function of concentration are used to calculate IC 50 values.

Article Snippet: Equal amounts of proteins were resolved using NuPAGE 4-12% Bis-Tris 15-well or 26-well gels (Invitrogen, #NP0336BOX or #WG1403BOX), transferred onto PVDF membranes (Thermo Fisher Scientific, iBlot 2 Gel Transfer Device, #IB21001), and immunoblotted with anti-IRAK1 (Cell Signaling, #4504, rabbit mAb; 1:1000 dilution), anti-phospho-IκBα (Ser32/36) (Cell Signaling, #9246; mouse mAb; 1:1000 dilution), anti-IκBα (Cell Signaling, #9242, rabbit pAb; 1:1000 dilution), anti-phospho-NFκB p65 (Ser536) (Cell Signaling, #3033, rabbit mAb; 1:1000 dilution), anti-NFκB p65 (Cell Signaling, #8242, rabbit mAb; 1:1000 dilution), and anti-β-actin (Cell Signaling, #3700, mouse mAb; 1:3000 dilution) antibodies, diluted in TBS-T (Boston BioProducts, Inc., #IBB-180X) containing 5% BSA, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Promega, #W4011 or #W4021), diluted 1:10000 in TBS-T containing 5% milk, and visualized using ECL western blotting substrates (Pierce ECL Western Blotting Substrate, #32106).

Techniques: Phospho-proteomics, Western Blot, Incubation, Binding Assay, Concentration Assay

Journal: bioRxiv

Article Title: A druggable redox switch on SHP1 controls macrophage inflammation

doi: 10.64898/2026.02.19.706790

Figure Lengend Snippet: a, LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation over a time course of 2 h in iBMDMs pre-treated with DMSO or SCA1 (50 μM) for 3 h. Immunoblots shown are representative of three independent experiments. b, LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation in iBMDMs following 3 h pre-incubation with DMSO, SCA1 or its non-covalent reversible analog of SCA1 (SCA1-NC) at the indicated concentrations. Immunoblots shown are representative of three independent experiments. c , SHP1 binding by SCA derivatives in iBMDMs treated at 5 μM followed by competition for SHP1 binding with biotinylated SCA1 (10 μM). d, Stoichiometric engagement score (R) of each cysteine quantified in iBMDM cysteine proteome treated with SCA9 (50 μM), showing SHP1 Cys102 as top hit exhibiting >50% engagement by SCA9. R = (S/N of DMSO) / (S/N of SCA9), where S/N is signal-to-noise ratio. e , LPS-induced (100 ng/ml) IκBα degradation and NF-κB p65 phosphorylation in iBMDMs following 3 h pre-incubation with DMSO or SCA derivatives at the indicated concentrations. Immunoblots shown are representative of two independent experiments. f , Pro-inflammatory cytokine IL-6 and TNF levels in cell supernatants of iBMDMs pre-incubated 3 h with SCA1, SCA1-NC or the respective derivatives over a dose response (0.313-80 μM) followed by 6 h LPS stimulation (100 ng/ml). Normalized percent cytokine production as a function of concentration are used to calculate IC 50 values.

Article Snippet: Equal amounts of proteins were resolved using NuPAGE 4-12% Bis-Tris 15-well or 26-well gels (Invitrogen, #NP0336BOX or #WG1403BOX), transferred onto PVDF membranes (Thermo Fisher Scientific, iBlot 2 Gel Transfer Device, #IB21001), and immunoblotted with anti-IRAK1 (Cell Signaling, #4504, rabbit mAb; 1:1000 dilution), anti-phospho-IκBα (Ser32/36) (Cell Signaling, #9246; mouse mAb; 1:1000 dilution), anti-IκBα (Cell Signaling, #9242, rabbit pAb; 1:1000 dilution), anti-phospho-NFκB p65 (Ser536) (Cell Signaling, #3033, rabbit mAb; 1:1000 dilution), anti-NFκB p65 (Cell Signaling, #8242, rabbit mAb; 1:1000 dilution), and anti-β-actin (Cell Signaling, #3700, mouse mAb; 1:3000 dilution) antibodies, diluted in TBS-T (Boston BioProducts, Inc., #IBB-180X) containing 5% BSA, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Promega, #W4011 or #W4021), diluted 1:10000 in TBS-T containing 5% milk, and visualized using ECL western blotting substrates (Pierce ECL Western Blotting Substrate, #32106).

Techniques: Phospho-proteomics, Western Blot, Incubation, Binding Assay, Concentration Assay