Journal: Frontiers in medicine

Article Title: ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

doi: 10.3389/fmed.2021.717602

Figure Lengend Snippet: FIGURE 1 | Localization of ROCKs in GFAP-positive cells in fibrovascular tissues of patients with PDR. (A) Representative fluorescent micrographs of ROCK1 immunofluorescence in fibrovascular tissues. (a) Green, GFAP (Alexa Fluor® 488). (b) Red, ROCK1 (Alexa Fluor® 546). (c) Blue, nuclei counterstained with DAPI. (d) Merged image. Arrows indicate the co-localization of ROCK1 with GFAP in the fibrovascular tissue. (e) High-magnification image. (f) Negative control (mouse and rabbit IgG) in sequential sections. (B) Representative fluorescence micrographs of ROCK2 immunofluorescence in fibrovascular tissues. (a) Green, GFAP (Alexa Fluor®

Article Snippet: Membranes were incubated with 5% skim milk for 1 h and then incubated with primary antibodies against ROCK1 (1:5000, EP786Y, Abcam), MLC2 (1:1000, #8505, Cell Signaling), p-MLC2 (1:1000, #3675, Cell Signaling), MYPT1 (1:1000, #2634, Cell Signaling), pMYPT1 (1:1000, #5163, Cell Signaling), and cleaved ROCK1 (1:1000, 154C1465, Novus biologicals, Centennial, CO, USA) at 4◦C overnight, followed by incubation with goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (1:4000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate Frontiers in Medicine | www.frontiersin.org 4 September 2021 | Volume 8 | Article 717602 (1:4000, Jackson Immunoresearch) at room temperature for 1 h. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: Negative Control

Journal: Frontiers in medicine

Article Title: ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

doi: 10.3389/fmed.2021.717602

Figure Lengend Snippet: FIGURE 2 | Effect of acrolein on transcriptional levels of Rocks, Mypt1, and Mlc2 in TR-MUL5 cells. The mRNA expression levels of (A) Rock1, (B) Rock2, (C) Mypt1, and (D) Mlc2 when TR-MUL5 cells were incubated with acrolein (0–50 µM, n = 6 each). Values represent mean ± SEM; *P < 0.05; **P < 0.01; N.S., not significant.

Article Snippet: Membranes were incubated with 5% skim milk for 1 h and then incubated with primary antibodies against ROCK1 (1:5000, EP786Y, Abcam), MLC2 (1:1000, #8505, Cell Signaling), p-MLC2 (1:1000, #3675, Cell Signaling), MYPT1 (1:1000, #2634, Cell Signaling), pMYPT1 (1:1000, #5163, Cell Signaling), and cleaved ROCK1 (1:1000, 154C1465, Novus biologicals, Centennial, CO, USA) at 4◦C overnight, followed by incubation with goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (1:4000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate Frontiers in Medicine | www.frontiersin.org 4 September 2021 | Volume 8 | Article 717602 (1:4000, Jackson Immunoresearch) at room temperature for 1 h. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: Expressing, Incubation

Journal: Frontiers in medicine

Article Title: ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

doi: 10.3389/fmed.2021.717602

Figure Lengend Snippet: FIGURE 3 | Effect of acrolein on protein levels of ROCK1, MYPT1, and MLC2 in TR-MUL5 cells. (A) Western blotting of total and cleaved forms of ROCK1 protein when TR-MUL5 cells were incubated with acrolein (0–50 µM). (B,C) Densitometric analysis of the bands. (B) Total forms of ROCK1 protein (0–50 µM, n = 3 each). (C) Relative cleaved forms of ROCK1 protein (0–50 µM, n = 4 each). (D) Total protein of MYPT1 and MLC2 in TR-MUL5 cells exposed to acrolein (0–50 µM). Values represent mean ± SEM; *P < 0.05.

Article Snippet: Membranes were incubated with 5% skim milk for 1 h and then incubated with primary antibodies against ROCK1 (1:5000, EP786Y, Abcam), MLC2 (1:1000, #8505, Cell Signaling), p-MLC2 (1:1000, #3675, Cell Signaling), MYPT1 (1:1000, #2634, Cell Signaling), pMYPT1 (1:1000, #5163, Cell Signaling), and cleaved ROCK1 (1:1000, 154C1465, Novus biologicals, Centennial, CO, USA) at 4◦C overnight, followed by incubation with goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (1:4000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate Frontiers in Medicine | www.frontiersin.org 4 September 2021 | Volume 8 | Article 717602 (1:4000, Jackson Immunoresearch) at room temperature for 1 h. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: Western Blot, Incubation

Journal: Frontiers in medicine

Article Title: ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.

doi: 10.3389/fmed.2021.717602

Figure Lengend Snippet: FIGURE 4 | Impact of acrolein stimulation on antioxidant defense systems in TR-MUL5 cells. (A) Total GSH levels when TR-MUL5 cells were exposed to acrolein for 3 h. (B) Rock1 mRNA levels in acrolein-stimulated TR-MUL5 cells with or without treatment of N-acetylcysteine (NAC) (n = 3 each). (C) Rock1 mRNA levels in TR-MUL5 cells treated with or without hydrogen peroxide (n = 3 each). Values represent mean ± SEM; *P < 0.05; **P < 0.01.

Article Snippet: Membranes were incubated with 5% skim milk for 1 h and then incubated with primary antibodies against ROCK1 (1:5000, EP786Y, Abcam), MLC2 (1:1000, #8505, Cell Signaling), p-MLC2 (1:1000, #3675, Cell Signaling), MYPT1 (1:1000, #2634, Cell Signaling), pMYPT1 (1:1000, #5163, Cell Signaling), and cleaved ROCK1 (1:1000, 154C1465, Novus biologicals, Centennial, CO, USA) at 4◦C overnight, followed by incubation with goat anti-mouse IgG (H+L) horseradish peroxidase conjugate (1:4000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate Frontiers in Medicine | www.frontiersin.org 4 September 2021 | Volume 8 | Article 717602 (1:4000, Jackson Immunoresearch) at room temperature for 1 h. Signals were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: