mouse anti sod3  (Santa Cruz Biotechnology)

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Low vascular EC-SOD exacerbates hypoxia-induced interstitial macrophage accumulation. Flow cytometric analysis of lung interstitial macrophage (IM) numbers in mice expressing low vascular EC-SOD (EC-SOD SMC KO; EC-SOD loxp/loxp × Tg cre/SMMHC ) compared with floxed wild-type controls (WT; EC-SOD loxp/loxp ) at baseline (normoxia, Nx) and following 4 or 14 days of hypoxia exposure (Hx). Intravenous (IV) CD45 antibody administration was used to exclude intravascular cells as part of the “Dump” gate (gating shown in ). Total macrophages were determined as live IV-CD3 − B220 − Ly6G − CD45 + CD64 + singlets. IMs (CD64 + , CD11b hi CD11c low/int ) and resident alveolar macrophages (AM; CD64 + , CD11b hi CD11c low/int ) were separated based on CD11b and CD11c expression and IM1 (CD11c low MHCII low ), IM2 (CD11c low MHCII hi ), and IM3 (CD11c int MHCII hi ) subsets identified by the expression of CD11c and MHCII. Total macrophages ( A ), interaction (hypoxia:genotype) P = 0.01480; resident AMs ( B ), interaction (hypoxia:genotype) P = 0.02250; total IMs ( C ), interaction (hypoxia:genotype) P = 0.04467; IM1( D ), interaction (hypoxia:genotype) P = 0.02789; IM2 ( E ), interaction (hypoxia: genotype) P = 0.06856; and IM3 ( F ) counts, interaction (hypoxia:genotype) P = 0.04813 in whole lung over the hypoxia time course. Two-factor aligned rank test ANOVA, Tukey’s post hoc tests for comparisons, all P values where P < 0.2 are shown. n = 10–15 mice per group. Due to the SMMHC promotor’s location on the Y chromosome, all mice are male. All data are nonparametric and expressed as median ± interquartile range. EC-SOD, extracellular superoxide dismutase; KO, knockout; SMC, smooth muscle cell; SMMHC, SMC myosin heavy chain.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Expressing, Knock-Out

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Single dose of SOD mimetic before hypoxia attenuates pulmonary hypertension and vascular remodeling. A : chronic hypoxic pulmonary hypertension was assessed by direct right ventricle (RV) puncture and measurement of RV systolic pressure (RVSP). B : RV hypertrophy was assessed by right ventricle/left ventricle + septum weights. Muscularization of small pulmonary vessels after 21 days of hypoxia was evaluated using α-smooth muscle actin (α-SMA) antibody staining (purple) of lung sections from hypoxic mice treated with SOD mimetic or vehicle (PBS) compared with normoxic controls. Costaining was performed with HA-BP to also detect hyaluronan (brown). C : vessel muscularization at 21 days was quantified by counting the number of muscularized small vessels (<50 μm) with positive staining for α-SMA counted per high-power field (HPF). D : representative images taken at ×10 magnification (scale bars represent 200 μm) with muscularized vessels indicated with black arrows. One-way ANOVA, Tukey’s post hoc tests for multiple comparisons, n = 5–9 mice per group. Female mice indicated with closed symbols. All data expressed as means ± SD. HA-BP, hyaluronan-binding protein; Hx, hypoxia; Nx, normoxia; SOD, superoxide dismutase.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Staining, Binding Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Perivascular Lyve1 + IMs are increased in EC-SOD SMC KO following 4 days of hypoxia and colocalize with HA. Lung sections were stained with a panel of antibodies to identify macrophage populations around pulmonary vessels in floxed WT controls and EC-SOD SMC KO mice at baseline (left hand side images) and during peak hypoxia-induced IM accumulation (4 D Hx; right hand side images). Immunofluorescence imaging was performed on the Vectra Polaris (Akoya) using inForm software. F480 was used to label all macrophages in white ( A ), CD163 in green ( B ), and Lyve1 in yellow ( C ) were used to label the Lyve1 + IM subset. α-SMA was used to identify smooth muscle (blue) in panels. Representative images showing increased interstitial macrophages in hypoxic WT and hypoxic EC-SOD SMC KO mice are shown from n = 3 mice per group; 40 magnification, scale bars 20 μm. White arrows indicate Lyve1-IMs and yellow arrows indicate Lyve1 + IMs in each section. D : adjacent lung sections were costained for α-SMA (purple) actin; EC-SOD, and HA-BP to detect perivascular hyaluronan (brown). Representative images taken at ×40 magnification (scale bars = 50 μm) from n = 3–5 mice per group. Due to the SMMHC promotor’s location on the Y chromosome, all mice used were male. α-SMA, α-smooth muscle actin; EC-SOD, extracellular superoxide dismutase; HA, hyaluronan; HA-BP, hyaluronan-binding protein; Hx, hypoxia; IMs, interstitial macrophages; KO, knockout; Lyve1, lymphatic vessel endothelial hyaluronan receptor 1; Nx, normoxia; SMC, smooth muscle cell; SMMHC, SMC myosin heavy chain; WT, wild type.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Staining, Immunofluorescence, Imaging, Software, Binding Assay, Knock-Out

Journal: bioRxiv

Article Title: Smooth muscle LRRC8A knockout preserves vascular function in AngII hypertension

doi: 10.1101/2025.05.08.652978

Figure Lengend Snippet: Antioxidant enzyme expression, proliferation and senescence are altered in abdominal aortae from LRRC8A KO mice. ( A ) CuZnSOD expression was not altered by LRRC8A KO or by AngII hypertension. ecSOD was increased by AngII only in KO vessels. Catalase and HO-1 expression were reduced in KO AngII hypertension compared to WT AngII. ( B ) iNOS and LRRC8A expression was enhanced by AngII hypertension in WT aortae. PCNA expression was increased in AngII hypertension ( p = 0.06), but this effect was reduced in KO vessels. ( C ) The senescence marker β-galactosidase (β-gal) increased by AngII hypertension in both WT and KO, but this effect was reduced in KO vessels. Data are mean ± SEM normalized to WT Sham. * p < 0.05 compared to WT Sham. † p < 0.05 (n=3 to 4).

Article Snippet: Antibodies were as follows: sGCα (#12605-1-AP), sGCβ (#19011-1-AP), cofilin (#66057-1-Ig) from Proteintech (Rosemont, IL), p-CPI-17 (#AB52174, Abcam, Cambridge, UK), CPI-17 (#sc-48406), ERM (#sc-271456), GAPDH (#sc-59540) from Santa Cruz Biotechnology (Dallas, TX), p-ERM (#3726), p-cofilin (#3313), Catalase (#14097), HO-1 (43966) from Cell Signaling Technology (Danvers, MA), CuZnSOD (#AF3418), ecSOD (#AF4817) from R&D systems (Minneapolis, MN), iNOS (#610328, BD Transduction, San Jose, CA), LRRC8A (#A304-175A, Bethy Laboratories, Montgomery, TX), PCNA (#05-347, MilliporeSigma, Burlington, MA), β-galactosidase (#GTX134513, GeneTex, Irvine, CA), Tubulin (Vanderbilt Antibody Core, Nashville, TN).

Techniques: Expressing, Marker