protein markers  (Bio-Rad)

Journal: Aging Cell

Article Title: NMNAT1 Activates Autophagy to Delay D‐Galactose‐Induced Aging in Cochlear Hair Cells

doi: 10.1111/acel.70373

Figure Lengend Snippet: Establishment of D‐gal‐treated aging model in HEI‐OC1 cells. (A) Schematic illustration of D‐gal treatment in HEI‐OC1 cells. (B) Cell viability after exposure to different concentrations of D‐gal, evaluated using the CCK‐8 assay ( n = 4, independent samples). (C) Immunofluorescent staining of Mito‐SOX in HEI‐OC1 cells exposed to D‐gal (5 and 20 mg/mL) for 72 h. (D) Quantitative analysis of Mito‐SOX intensity in C ( n = 4, independent samples). (E) Mito‐SOX levels were detected using flow cytometry in HEI‐OC1 cells exposed to D‐gal (5 and 20 mg/mL) for 72 h. (F) Statistical analysis of Mito‐SOX levels in E ( n = 3, independent samples). (G) Immunofluorescent staining of DCFH‐DA in HEI‐OC1 cells. (H) Quantitative analysis of DCFH‐DA intensity in G ( n = 4, independent samples). (I) DCFH‐DA levels were detected using flow cytometry in HEI‐OC1 cells. (J) Statistical analysis of DCFH‐DA levels in I ( n = 3, independent samples). (K–M) Western blot analysis of senescence marker protein‐30 (SMP30), Lamin B1, and γ‐H2A.X in HEI‐OC1 cells treated with D‐gal. (N–P) Statistical analysis of SMP30 ( n = 4, independent samples), Lamin B1 ( n = 3, independent samples), and γ‐H2A.X ( n = 3, independent samples) expression in K–M. Scale bar: 20 μm. Statistical significance as ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti‐β‐tubulin (1:40000, 5568; CST), rabbit anti‐β‐Actin (1:40000, 20536‐1‐AP; Proteintech), mouse anti‐GAPDH (1:4000, ab8245; Abcam), rabbit anti‐senescence marker protein‐30 (SMP30) (1:1000, 17947‐1‐AP; Proteintech), rabbit anti‐lamin B1 (1:1000, 13435; CST), rabbit anti‐γ‐H2A.X (1:400, 2577; CST), rabbit anti‐P21 (1:1000, 37543; CST), rabbit anti‐P16 (1:1000, 29271; CST), rabbit anti‐NMNAT1 (1:1000, 98354; CST), rabbit anti‐NMNAT1 (1:400, 11399‐1‐AP; Proteintech), rabbit anti‐LC3B (1:1000, 3868; CST), rabbit anti‐P62 (1:1000, 23214; CST), mouse anti‐autophagy related 7 (ATG7) (1:1000, 67341; Proteintech), and rabbit anti‐BECLIN1 (1:1000, 3495; CST).

Techniques: CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Marker, Expressing

Journal: Aging Cell

Article Title: NMNAT1 Activates Autophagy to Delay D‐Galactose‐Induced Aging in Cochlear Hair Cells

doi: 10.1111/acel.70373

Figure Lengend Snippet: Establishment of D‐gal‐induced aging model in cochlear explants. (A) Immunofluorescence staining demonstrating the number of myosin7a + hair cells in cochlear explants treated with 20, 30, and 40 mg/mL D‐gal for 72 h. (B) Quantification of the number of myosin7a + hair cells in cochlear explants ( n = 4; four individual explants from four mice). (C–E) Western blotting of senescence marker protein‐30 (SMP30), Lamin B1, and γ‐H2A.X expression in cochlear explants exposed to D‐gal. (F–H) Statistical analysis of SMP30 ( n = 5), Lamin B1 ( n = 3), and γ‐H2A.X ( n = 3) in C–E; n corresponds to the number of independent samples, each consisting of 12 explants from six mice. (I) Senescence‐associated β‐gal staining images of hair cells in cochlear explants exposed to D‐gal. (J) Quantification of β‐gal staining intensity in I ( n = 4; four individual explants from four mice). Scale bar: 20 μm. Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti‐β‐tubulin (1:40000, 5568; CST), rabbit anti‐β‐Actin (1:40000, 20536‐1‐AP; Proteintech), mouse anti‐GAPDH (1:4000, ab8245; Abcam), rabbit anti‐senescence marker protein‐30 (SMP30) (1:1000, 17947‐1‐AP; Proteintech), rabbit anti‐lamin B1 (1:1000, 13435; CST), rabbit anti‐γ‐H2A.X (1:400, 2577; CST), rabbit anti‐P21 (1:1000, 37543; CST), rabbit anti‐P16 (1:1000, 29271; CST), rabbit anti‐NMNAT1 (1:1000, 98354; CST), rabbit anti‐NMNAT1 (1:400, 11399‐1‐AP; Proteintech), rabbit anti‐LC3B (1:1000, 3868; CST), rabbit anti‐P62 (1:1000, 23214; CST), mouse anti‐autophagy related 7 (ATG7) (1:1000, 67341; Proteintech), and rabbit anti‐BECLIN1 (1:1000, 3495; CST).

Techniques: Immunofluorescence, Staining, Western Blot, Marker, Expressing