rabbit anti phospho hsp27  (R&D Systems)

Journal: NPJ Science of Food

Article Title: A small molecule strategy with forskolin and p38 inhibitor for serum-free muscle stem cell expansion

doi: 10.1038/s41538-026-00732-8

Figure Lengend Snippet: a Expansion fold per passage of bMuSCs cultured under the indicated culture conditions. b Total cell yield after three consecutive passages. c Representative images of EdU incorporation (red) with DAPI nuclear counterstaining (blue) in P5 cells cultured. Scale bar, 250 μm. d Quantification of EdU-positive cells at P5. e cAMP concentrations measured in P5 cells. f Representative Western blot images showing p-HSP27 protein expression, with GAPDH used as a loading control. g Densitometric quantification of relative p-HSP27 protein levels in P5 cells, normalized to GAPDH. Data are presented as mean ± SEM from three independent experiments performed using cells derived from the same donor ( n = 3). Statistical significance was determined by comparison with Beefy-9: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Subsequently, the membranes were blocked with 1× TBST solution containing 5% nonfat milk for 2 h and incubated overnight at 4 °C with primary antibodies: p-HSP27 (CST, Cat# 9709), Pax7 (Thermo, Cat# PA5-68506), Pax3 (Affinity, Cat# DF6548), Myf5 (absin, Cat# abs146209), MyoD (CST, Cat# 13812), MyoG (BD, Cat# 556358), MyHC (Abcam, Cat# ab37484), Collagen I (Abcam, Cat# ab37484), GAPDH (Millipore, Cat# MAB374).

Techniques: Cell Culture, Western Blot, Expressing, Control, Derivative Assay, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: MK5 Regulates Microglial Activation and Neuroinflammation in Experimental Stroke Models

doi: 10.1111/cns.70395

Figure Lengend Snippet: MK5 gene knockout in microglia may participate in neuroinflammatory responses by downregulating HSP27 to mediate the NF‐κB signaling pathway. (A) Western blot analysis of HSP27, pHSP27, IκB, pIκB, NF‐κB, pNF‐κB protein expression in the peri‐infarct cortex of MK5 f/f and MK5 cKO mice 3 days post‐MCAO. (B–D) Statistical analysis of the pHSP27/HSP27, pIκB/IκB, and pNF‐κB/NF‐κB ratio. n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Western blot analysis of HSP27, pHSP27, IκB, pIκB, NF‐κB, pNF‐κB protein expression in BV2 cells transfected with Ctrl or si‐MK5 and subjected to OGD/R. (F–H) Statistical analysis of the pHSP27/HSP27, pIκB/IκB, and pNF‐κB/NF‐κB ratio. n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies were as follows: MK5 (1:1000, mouse; sc‐46667, Santa Cruz, USA), Iba‐1 (1:1000, rabbit; 17198, CST, USA), IκB (1:1000, rabbit; 4812, CST, USA), phospho‐IκB (1:1000, rabbit; 2859, CST, USA), HSP27 (1:1000, rabbit; 2442, CST, USA), phospho‐ HSP27 (1:1000, rabbit; 9709, CST, USA), Arginase‐1 (1:1000, rabbit; 89872, CST, USA), CD206 (1:1000, rabbit; 24595, CST, USA), IL‐1β (1:1000, rabbit; 12426, CST, USA), TNF‐α (1:1000, rabbit; 3707, CST, USA), NF‐κB (1:1000, rabbit; 8242, CST, USA), phospho‐NF‐κB (1:1000, rabbit; 3033, CST, USA), and GAPDH (1:1000, mouse; 60004‐1‐Ig, Protein Tech, Wuhan, China).

Techniques: Gene Knockout, Western Blot, Expressing, Transfection

Journal: bioRxiv

Article Title: Cellular mechanisms of early brain overgrowth in autistic children: elevated levels of GPX4 and resistance to ferroptosis

doi: 10.1101/2025.01.30.635706

Figure Lengend Snippet: (A) Representative flow cytometric histograms of ferrous iron (Fe 2+ ) accumulation measured using a Far-red Labile Fe 2+ Dye in TD-N, ASD-N, and ASD-DM NPCs. (B) Quantification of the mean fluorescence intensity of Fe 2+ accumulation in NPCs from TD-N, ASD-N, and ASD-DM NPCs. (C) Representative flow cytometric histograms of cellular reactive oxygen species (cROS) measured using a CELLROX Deep Red reagent in TD-N, ASD-N, and ASD-DM NPCs. (D) Quantification of the mean fluorescence intensity of cROS levels in TD-N, ASD-N, and ASD-DM NPCs. (E) Western blot analysis of p-HSP27 and GPX4 proteins in TD-N, ASD-N, and ASD-DM NPCs. GAPDH serves as a loading control. (F) Quantification of protein expression levels of p-HSP27 and GPX4 in TD-N, ASD-N, and ASD-DM NPCs, normalized to GAPDH levels. (G) Quantification of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) using a Luminescent-Based Assay in TD-N, ASD-N, and ASD-DM NPCs. (H) Quantification of the percentage of viable NPCs from TD-N, ASD-N, and ASD-DM conditions after 48h treatment with FIN56 at increasing doses (0, 0.1, 0.5, 1, 2.5, 5 μM), measured using the MTS assay. Data are expressed relative to the corresponding untreated cell lines. (I) Representative electron microscopic images of TD-N, ASD-N, and ASD-DM NPCs. TD-N NPCs treated with 5 μM FIN56 for 20h were used as a positive control for ferroptosis, and the TD-N NPCs treated with 1 μM staurosporine for 3h were used as a positive control for apoptosis. Top row scale bars, 2 μm. Bottom row scale bars, 600 nm. White arrows: shrunken mitochondria. Red arrows: chromatin condensation and stress granules. (J) Quantification of mitochondrial area of 5 mitochondria per cell for a total of 10 cells from each sample (total of 50 mitochondria per cell line; 200 mitochondria per condition). (K) Quantification of mitochondrial cristae area of 5 mitochondria per cell for a total of 10 cells from each sample (total of 50 mitochondria per cell line; 200 mitochondria per condition). Individual crista areas were summed up for the total cristae surface area of each mitochondrion. (L) All data are presented as mean ± standard deviation (*p ≤ 0.05; **p < 0.01; ***p < 0.001; **** p <0.0001). See also Figure S5, S6, and S7.

Article Snippet: The primary antibodies assessed include: Rabbit-anti-PAX6 (1:2000; BioLegend); Apoptosis Western Blot Cocktail (1:250; Abcam); Rabbit-anti-phospho-HSP27 (1:1000; R&D Systems); Rabbit-anti-GPX4 (1:1000; Cell Signaling); Rabbit-anti-gamma H2A.X (1:100000; Abcam); Necroptosis antibody sampler kit (1:1000; Cell Signaling); Rabbit-anti-GAPDH (1:1000; Cell Signaling) and Rabbit-anti-β-actin (1:1000; Cell Signaling).

Techniques: Fluorescence, Western Blot, Control, Expressing, Luminescence Assay, MTS Assay, Positive Control, Standard Deviation