Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: IL‐17A NAb inhibited EMT in SHR renal tissues. (A) Representative IHC images showing the expression of E‐cadherin, α‐SMA, and Collagen III, with quantitative analysis of their positive areas. (B) Representative immunoblots and relative expression levels of E‐cadherin, α‐SMA, and Collagen III proteins. (C) mRNA expression levels of E‐cadherin, α‐SMA, and Collagen III. Data are expressed as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Expressing, Western Blot

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 1. ClockΔ19/+ (Clk/+) mutant mice displayed improved glucose homeostasis and resistance to period lengthening and BMAL1 depletion under high-fat diet (HFD) feeding. Fasting glucose (a) serum insulin (b) glucose tolerance (c) and insulin tolerance (d) in WT and Clk/+ mutant mice after HFD feeding for 12 weeks (n = 9 − 11). For (c) and (d) area under curve (A.U.C) comparison is shown next to the graphs. (e) Representative actograms in each condition were shown. The mice were kept on a 12 h:12 h light:dark cycle (represented in the bar above) for 16 days and then released into constant darkness. (f) Circadian free-running period (tau) was calculated by using the ActiView software (n = 3 − 5). (g) Liver tissues were collected over the circadian time course from mice fed with regular chow (RC) or high-fat diet (HFD). Lysates were prepared and subjected to immunoblotting analysis using antibodies against clock proteins and GAPDH as control. The results were representative of three independent experiments. *p < 0.05, **p < 0.01, one-way ANOVA.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Mutagenesis, Comparison, Software, Western Blot, Control

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 2. CLOCK and CLOCKΔ19 exerted differential effects on BMAL1 ubiquitination and stability. (a,b) Protein lysates and total RNAs were prepared from wild-type (WT), ClockΔ19/+ (Clk/+ ) and ClockΔ19/Δ19 (Clk/Clk) MEF cells and subjected to Western blot analysis of CLOCK and BMAL1 (a) and real-time qPCR analysis of Bmal1 transcript levels (b). GAPDH was used as a loading control in (a). (c) WT, Clk/+ and Clk/ Clk MEF cells were exposed to cycloheximide (CHX; 25 μ g/ml) for the indicated times, and BMAL1 levels were assessed by immunoblotting analysis. Quantification is presented in the lower panel. Error bars represent mean ± SD (n = 3). Half-life was determined by using the GraphPad Prism software with nonlinear, one-phase exponential decay analysis. The analysis calculated the half-life parameter K at 0.191/h, 0.050/h, and 0.054/h in WT, Clk/+ and Clk/Clk MEF cells, corresponding to half-life at 3.63 h, 14.0 h and 12.9 h respectively. The K values are significantly different among all three conditions: p < 0.0001. Half-life was determined by using nonlinear, one-phase exponential decay analysis. Half-life parameter, K, is significantly different in all three conditions: p < 0.0001. (d) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK and/ or Flag-CLOCKΔ 19 were subjected to immunoblotting analysis with antibodies against the Flag-tag and GAPDH (loading control). Quantitation of three independent experiments is presented in the lower panel. (e) Cytoplasmic and nuclear fractions of 293T cells transfected with the indicated expression vectors were subjected to immunoblotting analysis with antibodies against anti-Flag, α -Tubulin and Lamin B1. The data represent the mean ± SD of three independent experiments. (f) 293T cells transfected with the indicated expression vectors for Flag-BMAL1, His-tagged ubiquitin (His-Ub), Flag-CLOCK, and Flag-CLOCKΔ 19 were lysed and immunoprecipitated with anti-Flag antibody prior to immunoblotting analysis with anti-BMAL1 and -CLOCK antibodies. The relative levels of the targeted proteins were measured by densitometry using the Image J program and the ratios were calculated relative to the GAPDH control.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Ubiquitin Proteomics, Western Blot, Control, Software, Transfection, Expressing, FLAG-tag, Quantitation Assay, Immunoprecipitation

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 3. CLOCKΔ19 attenuated BMAL1 degradation via both proteasomal and autophagic pathways. (a) WT, Clk/+ , and Clk/Clk MEF cells were exposed to 10 μ M MG132, 5 mM 3-methyladenine (3-MA) or 25 μ M chloroquine (CQ) for 4, 24 and 4 h, respectively. Cell lysates were subjected to immunoblotting analysis with antibodies against BMAL1, CLOCK and GAPDH (loading control). Representative results from three independent experiments are shown. (b) 293T cells transfected with the indicated expression vectors were treated with MG132 and 3-MA for 4 and 24 h followed by CHX exposure for the indicated times, and subjected to immunoblotting with antibodies against Flag and GAPDH (loading control). (c–e) 293T cells transfected with the indicated expression vectors were incubated in the absence or presence of MG132 (10 μ M) (c), 3-MA (1 mM) (d) or CQ (25 μ M) (e) for 4, 24, and 24 h, respectively. Lysates were subjected to immunoblotting analysis with antibodies against Flag and GAPDH (loading control).

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Western Blot, Control, Transfection, Expressing, Incubation

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 4. CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. (a,b) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag- CLOCKΔ 19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). (c) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 Δ UBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ 19, and Myc-p62 (WT or Δ UBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). (d) Protein lysates were prepared from p62+/+ (WT) and p62−/− MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. (e) MEFs (p62+/+ or p62−/−) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). (f) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 siRNA for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Transfection, Expressing, Western Blot, Control, Construct, Staining, Membrane, Negative Control