alpha b crystallin  (Proteintech)

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the methylation of CRYAB in vitro which exerted anti‐inflammatory effects. (A) BSP was performed to detect CRYAB methylation (white represented unmethylation, black represented methylation). (B) In rat astrocytes, Western blot was applied to check the changes of key factors in DNA methylation (DNMT1, DNMT3a, and DNMT3b) ( n = 3). (C) In rat astrocytes, the positive cells of CRYAB were checked using immunofluorescence. (D) In rat astrocytes, CRYAB protein expression was checked using Western blot ( n = 3). (E) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (F) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (G) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). Data in (B, D, E, F, and G) were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Methylation, In Vitro, Western Blot, DNA Methylation Assay, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Immunity, Inflammation and Disease

Article Title: MAFG Induces the Methylation of CRYAB to Promote the Activation of A1 Astrocyte After Spinal Cord Injury

doi: 10.1002/iid3.70334

Figure Lengend Snippet: Silencing of MAFG inhibited the activation of A1 astrocyte and neuroinflammation via CRYAB methylation. (A and B) In rat astrocytes, the mRNA expression of Serping1, C3, Sphk1, and S100A10 was checked by RT‐PCR ( n = 3). (C and D) In rat astrocytes, the protein expression of Serping1, C3, Sphk1, and S100A10 was checked using Western blot ( n = 3). (E and F) In supernatant of rat astrocytes, the levels of IL‐1β and IL‐6 were examined by ELISA ( n = 3). All data were analyzed using one‑way ANOVA followed by Tukey's post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: After blocking in 5% non‐fat milk for 1 h, membranes were incubated with primary antibodies against MAFG, C3, CRYAB, Serping1, DNMT3a (Abcam); S100A10, TNF‐α, IL‐1β (Proteintech); Sphk1, IL‐6, DNMT3b (Santa Cruz Biotechnology, USA); β‐actin and DNMT1 (Cell Signaling Technology, USA) overnight at 4°C, followed by incubation with the secondary antibodies (Abcam) for 1 h. To measure the optical density of protein bands, ImageJ software (NIH, USA) was used.

Techniques: Activation Assay, Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay