cytoplasmic and nuclear protein extraction kit  (Boster Bio)

Journal: Molecular Medicine

Article Title: OTULIN is a new target of EA treatment in the alleviation of brain injury and glial cell activation via suppression of the NF-κB signalling pathway in acute ischaemic stroke rats

doi: 10.1186/s10020-021-00297-0

Figure Lengend Snippet: EA inhibited the NF-κB signalling pathway via the upregulation of OTULIN expression. a Western blotting was used to detect OTULIN, p-IκBα, IκBα, and cytoplasm/nucleus-p65 proteins. β-Actin served as a loading control for total or cytoplasmic protein, and Lamin B served as a nuclear protein. b , n = 3) Quantitative analysis of OTULIN protein, the phosphorylation ratio of IκBα, and nuclear/cytoplasmic NF-κB p65. c , n = 6) Immunohistochemistry was performed to detect IκBα and NF-κB p65 proteins. Red arrows indicate IκBα- or NF-κB p65-positive cells. Scale bar = 100 μm. P

Article Snippet: The nuclear and cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (no. AR0106, Boster, Beijing, China), and the protein concentrations were detected using a BAC kit (Beyotime, Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Neuroscience

Article Title: Neuroprotective Effects of Celastrol on Transient Global Cerebral Ischemia Rats via Regulating HMGB1/NF-κB Signaling Pathway

doi: 10.3389/fnins.2020.00847

Figure Lengend Snippet: Schematic diagram illustrating possible neuroprotective mechanisms of celastrol against glial activation, oxidative stress and neuroinflammation in tGCI/R rats via the high mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) /nuclear factor (NF-κB) pathway. Cerebral ischemia induced HMGB1 nuclear-cytoplasmic translocation and activate TLR4/RAGE, promote the phosphorylation of IκBα, thus inducing the translocation of NF-κB from the cytoplasm to the nucleus, promote gene expression and generate a series of cytokines, thus promoting glial activation, neuroinflammation, oxidative stress, and caspase activation, eventually lead to apoptotic neuronal death. GFAP, glial fibrillary acid protein; Iba-1, ionized calcium binding adaptor molecular 1; ROS, reactive oxygen species; MDA, malondialdehyde; TNF-α, tumor necrosis factor α; IL-β, interleukin-β; IL-6, interleukin-6; IL-10, interleukin-10.

Article Snippet: Immunofluorescent Staining Three days (72 h) after tGCI/R, rat brains (n = 4) were freshly removed and divided into two halves with a sharp blade on ice, one half was used for immunofluorescent analysis and the other was used for nuclear/cytoplasmic protein extraction.

Techniques: Activation Assay, Translocation Assay, Expressing, Binding Assay, Multiple Displacement Amplification

Journal: The EMBO Journal

Article Title: Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling

doi: 10.15252/embj.2019103255

Figure Lengend Snippet: Deletion of Islr in stromal cells suppressed epithelial Yap activity A Heatmap of the altered Hippo‐related genes in colons from control and cKO mice 1 day after 5‐day DSS treatment. The parameter of the color key indicated the fold changes converted to log2 of signal value normalized. B qRT–PCR analysis validates the altered Hippo‐related genes in control and cKO mice; n = 4. C Western blotting for pMst1/2, Mst1, pMob1, and Mob1 in colon epithelium from control and cKO mice 2 days after 5‐day DSS treatment. β‐actin was used as a loading control. The quantification of pMst1/2 versus Mst1 and pMob1 versus Mob1 was shown under the corresponding protein bands. D Western blotting for Yap1 in nuclear and cytoplasmic proteins isolated from intestinal epithelial cells from control and cKO mice 2 days after 5‐day DSS treatment. Histone H3, a positive control for nuclear proteins. α‐Tubulin, a positive control for cytoplasmic proteins. The quantification of Yap1 versus Histone H3 in nuclear proteins was shown under the corresponding band. E, F Double immunofluorescence for Yap1 and β‐catenin in the colons from control ( n = 4) and cKO ( n = 4) mice 2 days after DSS removal (E), and in AOM/DSS tumors from control ( n = 4) and cKO ( n = 4) mice (F). The percentage of nuclear Yap1 + cells versus epithelial cells was quantified. Scale bar: 50 μm. G Western blotting for Yap1 and pYap1 in colon epithelial cells from control and cKO mice 2 days after 5‐day DSS treatment. β‐actin was used as a loading control. The ratio of Yap1/pYap1 was quantified; n = 3. H The growth curve of HCT116 colorectal cancer cells cultured in the supernatant from WT or cKO IMCs, concomitantly transfected with PCDH empty vector or PCDH‐YAP1‐5SA vector; n = 4 technical replicates. I Pearson correlation analysis of ISLR and CTGF ( P

Article Snippet: Nucleoprotein extraction Nucleoprotein was isolated from cells using a nucleoprotein extraction kit (AR0106, BosterBio, USA) according to the manufacturer's instructions.

Techniques: Activity Assay, Mouse Assay, Quantitative RT-PCR, Western Blot, Isolation, Positive Control, Immunofluorescence, Cell Culture, Transfection, Plasmid Preparation