Journal: Journal of Plant Physiology

Article Title: The enzymatic hydrolysate of fucoidan from Sargassum hemiphyllum triggers immunity in plants

doi: 10.1016/j.jplph.2023.153967

Figure Lengend Snippet: FEH induces MPK3/6 activation in Arabidopsis. Seven-day-old seedlings were treated with 400 μg/mL of FEH for 0, 15, 30, 60, and 150 min. MAPK activation was detected by immunoblotting with anti-pErk1/2 antibodies. GAPDH was detected by immunoblotting as the loading control.

Article Snippet: Total plant proteins were isolated from whole seedlings and the MAPK activation was examined by immunoblotting with anti-pErk1/2 antibodies (1/5000 dilution, Cell Signaling Technology, USA).

Techniques: Activation Assay, Western Blot

Journal: Journal of Cell Science

Article Title: Lamin A/C phosphorylation at serine 22 is a conserved heat shock response to regulate nuclear adaptation during stress

doi: 10.1242/jcs.259788

Figure Lengend Snippet: The effect of kinase inhibition on lamin A/C phosphorylation under HS. (A) Immunoblots showing lamin A/C, pSer22 lamin A/C, HSF1, ERK1/2, pERK1/2, AKT, pAKT, caspase-3 and cleaved caspase-3 protein levels in control and heat-shocked HeLa cells treated with different kinase inhibitors. GAPDH was used as a loading control and HeLa cells treated with 2 µM staurosporine (STA) as a positive control for apoptotic cell death. The average numerical values of signal intensities relative to the loading control are shown (GAPDH and lamin A/C, n =5). (B) Quantification of pSer22 lamin A/C WB intensity change (%) in control cells and cells treated with kinase inhibitors after 4 h HS at 42°C ( n =5). * P <0.05; ** P <0.01 (Mann–Whitney test). (C) Confocal microscopy images of LMNA WT/WT and LMNA WT/S143P fibroblasts stained for lamin A/C (green) and pSer22 lamin A/C (red) at normal culture conditions and after 2 h HS at 44°C with or without STA treatment. Scale bars: 5 µm. (D,E) Corrected total cell fluorescence (CTCF) of average pSer22 lamin A/C intensity values under HS and STA treatment ( n =50, from two biological replicates). *** P <0.001; §, compared to untreated 37°C; #, compared to STA 37°C (Kruskal–Wallis/Dunn’s test). (F,G) Nuclear area (F) and sphericity (G) were determined from LMNA WT/WT and LMNA WT/S143P fibroblasts after treatment with STA and exposure to 44°C for 1–2 h ( n =50, from two biological replicates). * P <0.05 (two-way ANOVA with Tukey’s post hoc test). Boxplots show the 75th, 50th and 25th percentiles, and the whiskers show the 95% c.i. for median.

Article Snippet: The primary antibodies used were: mouse monoclonal anti-lamin A/C (1:10,000, 5G4, kindly provided by Prof. Robert D. Goldman, Northwestern University, IL), anti-lamin B1 (1:1000, ab16048, Abcam) rabbit polyclonal anti-heat shock factor 1 (1:1000, ADI-SPA-901, Enzo Life Sciences, Farmingdale, NY), rat monoclonal anti-heat shock factor 1 (1:1000, 10H8, StressMarq Biosciences, Victoria, Canada), rabbit monoclonal anti-phospho-lamin A/C serine 22 (1:1000, D2B2E, Cell Signaling Technology), rabbit polyclonal anti-phospho-lamin A/C serine 392 (1:1000, ab58528, Abcam), rabbit polyclonal anti-AKT (pan) (1:1000, C67E7, Cell Signaling Technology), rabbit monoclonal anti-pAKT T303 (1:1000, D6F8, Cell Signaling Technology), rabbit polyclonal anti-ERK1 (1:500; K-23, Santa Cruz Biotechnology), rabbit polyclonal anti-ERK2 (1:500, c-14, Santa Cruz Biotechnology), rabbit monoclonal anti-pERK1/2 (1:1000, D13.14.4E, Cell Signaling Technology), rabbit monoclonal anti-cleaved PARP-1 (1:1000, E-51, Abcam), rabbit monoclonal anti-caspase-3 (1:1000, 8G10, Cell Signaling Technology), rabbit monoclonal anti-cleaved caspase-3 (1:1000, Asp175, Cell Signaling Technology), rabbit monoclonal anti-Lap2α (1:5000, 245/2, kindly provided by Prof. Roland Foisner, University of Vienna), mouse monoclonal anti-HSP70/HSP72 (1:1000, C92F3A-5, Enzo Life Sciences), rat monoclonal anti-HSC70/HSP73 (1:1000, 1B5, Enzo Life Sciences), HRP-conjugated anti-GAPDH (1:5000, ab9385, Abcam) and mouse monoclonal anti-actin (1:1000, AC-40, Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Positive Control, MANN-WHITNEY, Confocal Microscopy, Staining, Fluorescence