Journal: Neural Regeneration Research

Article Title: Salsolinol as an RNA m 6 A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

doi: 10.4103/NRR.NRR-D-23-01592

Figure Lengend Snippet: Autophagy regulated by Sal is partly related to the inhibition of YAP1. (A) Sal treatment decreased the expression of p62 and increased LC3. (B) Autophagy-related proteins were examined in cells treated with Sal alone or in combination with BafA1 treatment. (C) In PC12 cells with YAP1 knockdown, the levels of p62 decreased and LC3 increased. Data are expressed as the mean ± SD ( n = 3/group). * P < 0.05, *** P < 0.001, vs. NC group (Student’s t -test). (D) In PC12 cells treated with Verteporfin, the levels of p62 decreased and LC3 increased. (E) A schematic model in which Sal acts as an RNA m 6 A methylation inducer, mediating dopaminergic neuronal death by regulating YAP1 and autophagy. ALKBH5: alkB homolog 5; BafA1: Bafilomycin A1; FTO: fat mass and obesity-associated protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LC3: light chain 3; m 6 A: N 6 -methyladenosine; METTL3: methyltransferase-like 3; NC: normal control; p62: Sequestosome 1; Sal: salsolinol; siYAP1: small interfering RNA of YAP1; YAP1: Yes-associated protein 1; YTHDF2: YTH domain-containing family protein 2.

Article Snippet: The membrane was blocked with 5% skimmed milk and incubated with mouse anti-FTO monoclonal antibody (1:1000, Abcam, Cat# ab92821, RRID: AB_10565042), rabbit anti-ALKBH5 monoclonal antibody (1:1000, Abcam, Cat# ab195377, RRID: AB_2827986), rabbit anti-METTL3 monoclonal antibody (1:1000, Abcam, Cat# ab195352, RRID: AB_2721254), mouse anti-YAP1 monoclonal antibody (1:8000, Proteintech, Chicago, IL, USA, Cat# 66900-1-Ig, RRID: AB_2882229), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:100,000, Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), rabbit anti-YTH domain-containing family protein 1 (YTHDF1) polyclonal antibody (1:2000, Proteintech, Cat# 17479-1-AP, RRID: AB_2217473), rabbit anti-YTH domain-containing family protein 2 (YTHDF2) polyclonal antibody (1:5000, Proteintech, Cat# 24744-1-AP, RRID: AB_2687435), mouse anti-YTH domain-containing family protein 3 (YTHDF3) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, CA, USA, Cat# sc-377119, RRID: AB_2687436), rabbit anti-p62 polyclonal antibody (1:1000, MBL, Tokyo, Japan, Cat# PM045, RRID: AB_1279301), rabbit anti-light chain 3 beta (LC3B) polyclonal antibody (1:1000, Cell Signaling Technology, Boston, MA, USA, Cat# 2775, RRID:AB_915950), and rabbit anti-phospho-YAP1 (Ser127) (1:1000, Cell Signaling Technology, Cat# 13008, RRID: AB_2650553) at 4°C overnight.

Techniques: Inhibition, Expressing, Knockdown, Methylation, Control, Small Interfering RNA

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants

doi: 10.1007/s00018-023-05059-1

Figure Lengend Snippet: P5-P4′ variants are normally secreted by HEK293T/17 cells and do not polymerize. Supernatants of HEK293T/17 cells transiently transfected with expression vectors for M, Z, P5-P4′, and Iners AAT variants were collected 24 h post-transfection. Empty vector (mock) was included as negative control. a Sandwich ELISA quantification of total AAT in cell supernatants utilizing polyclonal antibodies for AAT capture and detection, normalized to the respective total protein content. Values are expressed relative to M-AAT (n = 3). b Western blot analysis of AAT content in equal volumes of cell supernatants resolved by either non-denaturing-PAGE (top panels) or reducing SDS-PAGE (bottom panels) and detected by a polyclonal anti-AAT antibody. Black and white arrowheads indicate the monomeric AAT form resolved under non-denaturing and denaturing/reducing conditions, respectively. Differences in the migration of the monomers of different RCL variants under non-denaturing conditions is due to altered charge. The blots are representative of three independent experiments. c Sandwich ELISA quantification of AAT polymers in cell supernatants, utilizing an AAT polymer-specific monoclonal antibody (2C1 mAb) as capture and polyclonal anti-AAT antibody for detection. Polymeric AAT is expressed as percentage of total AAT measured in panel (a) (n = 2). Data in a and c are reported as mean ± SD. Significant differences with M-AAT were identified with one-way ANOVA analysis corrected by Dunnett’s test (no asterisk: non-significant; ***p ≤ 0.01). The supernatants whose total AAT or polymeric AAT were below detection limit of the ELISA assays are reported as N.D

Article Snippet: AAT was detected by incubating with 2 μg/mL rabbit polyclonal antibody anti-AAT (Dako, A001202), overnight at room temperature, followed by incubation with 100 ng/mL HRP-conjugated donkey antibody anti-rabbit IgG (Cytiva, NA9340) in TBS-T, for 2 h at room temperature.

Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Sandwich ELISA, Western Blot, SDS Page, Migration, Polymer, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants

doi: 10.1007/s00018-023-05059-1

Figure Lengend Snippet: Complex formation between NE and secreted AAT variants. Each secreted AAT variant (20 nM) was incubated with increasing quantities of human NE for 1 h at room temperature, generating reactions at different NE:AAT molar ratios as shown. Reaction mixtures were resolved by SDS-PAGE under reducing conditions and AAT was detected with an anti-AAT polyclonal antibody. The AAT variants are grouped according to their behavior as a complex-forming, b substrate-like and c binding-deficient variants. Bands corresponding to AAT and AAT–NE complex are indicated by white and black arrowheads, respectively. Black arrows indicate the cleaved form of AAT that lacks the C-terminal fragment (S359-K394). Blots are representative of three independent experiments

Article Snippet: AAT was detected by incubating with 2 μg/mL rabbit polyclonal antibody anti-AAT (Dako, A001202), overnight at room temperature, followed by incubation with 100 ng/mL HRP-conjugated donkey antibody anti-rabbit IgG (Cytiva, NA9340) in TBS-T, for 2 h at room temperature.

Techniques: Variant Assay, Incubation, SDS Page, Binding Assay