Journal: International Journal of Molecular Sciences

Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

doi: 10.3390/ijms21186920

Figure Lengend Snippet: Effect of Bmal1 expression knockdown on adipocyte lipolysis in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were transfected with control siRNA or specific siRNA for Bmal1 using a transfection reagent. After 48 h, basal or isoproterenol-stimulated glycerol release was measured, and the cells were harvested for subsequent analysis by quantitative real-time PCR or Western blotting. ( A ) Relative expression levels of Bmal1 mRNA. The mRNA levels were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( B ) Representative immunoblotting data (upper panel) and relative densities of BMAL1 protein (bottom panel) measured by Western blotting, normalized to β-actin. ( C ) Expression levels of Hsl and ATGL mRNA were measured by quantitative real-time PCR using specific primers and normalized to the amount of 18S rRNA. ( D ) Representative immunoblotting data (upper panel) and relative densities of the HSL, ATGL, and Perilipin1 proteins (lower panel) measured by Western blotting and normalized to β-actin. ( E ) Representative immunoblotting data (upper panel) and relative densities of AKAP150 protein (bottom panel) measured by Western blotting, and normalized to β-actin. ( F ) Representative immunoblotting data of PKA-RIα, -RIIα, -RIIβ, and β-actin proteins. ( G ) Relative densities of the PKA- RIα, -RIIα, and -RIIβ proteins measured by Western blotting, and normalized to β-actin. The values in the bar graphs in panels A, B, C, D, E, and G are shown relative to the mean levels detected after treatment with control siRNA, which was arbitrarily set to 1. ( H ) Reduced response of lipolysis to isoproterenol in Bmal1 -siRNA adipocytes. ( I ) Representative immunoblotting data of HSL phosphorylated at Ser563 and Ser660. ( J , K ) Relative expression levels of HSL phosphorylated at Ser563 and Ser660 as measured by Western blotting and normalized to total HSL levels. The values in the bar graphs in panels H, J, and K are shown relative to the mean level detected in basal conditions, which was arbitrarily set to 1. In all experiments, values represent the means ± S.E. of three independent experiments. * p < 0.05, control-siRNA vs. Bmal1 -siRNA or basal vs. isoproterenol; intergroup differences; † p < 0.05, isoproterenol stimulation of control siRNA vs. that of Bmal1 siRNA.

Article Snippet: After separation by 6–12.5% SDS-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (ATTO), blocked for 60 min with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim milk, or with Block Ace ® Powder (DSP, Osaka, Japan) dissolved in purified water, and were then probed at 4 °C overnight in Can Get Signal @ Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan) against total HSL (Abcam, Cambridge, UK), HSL phosphorylated at Ser-563 (Cell Signaling Technology [CST], Danvers, MA, USA), HSL phosphorylated at Ser-660 (CST), ATGL (CST), total perilipin 1 (Abcam), CGI-58 (Santa Cruz Biotechnology, Dallas, TX, USA), PPARγ2 (ThermoFisher Scientific, Waltham, MA, USA), BMAL1 (Abcam), AKAP150 (Alomone Labs, Jerusalem, Israel), PKARIα (BD biosciences, Franklin Lakes, NJ, USA), PKARIIα (Abcam), PKARIIβ (Abcam), and β-actin (Abcam).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

doi: 10.3390/ijms21186920

Figure Lengend Snippet: Effect of exercise training timing on the levels of lipolysis-associated proteins in primary adipocytes isolated from epididymal adipose tissues. ( A ) Representative immunoblotting data and the relative amounts of ( B ) HSL, ( C ) ATGL, ( D ) perilipin1, ( E ) CGI-58, and ( F ) PPARγ2 protein, in primary isolated adipocytes from rats, measured by Western blotting and normalized to the amount of β-actin. ( G ) Relative protein expression levels of each band in the E-EX and L-EX groups (levels in respective sedentary controls were set to 1). ( H ) Representative immunoblotting data and the relative levels of ( I ) AKAP150, ( J ) PKA-RIα, ( K ) PKA-RIIα, and ( L ) PKA-RIIβ protein measured by Western blotting and normalized to the level of β-actin. ( M ) Bmal1 mRNA expression level measured by quantitative real-time PCR and normalized to the level of 18S rRNA. In all experiments, data are presented as the means ± S.E. ( n = 6). * p < 0.05, SED group vs. EX group. † p < 0.05, E-EX vs. L-EX. The abbreviations for the groups of rats are shown in and .

Article Snippet: After separation by 6–12.5% SDS-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (ATTO), blocked for 60 min with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim milk, or with Block Ace ® Powder (DSP, Osaka, Japan) dissolved in purified water, and were then probed at 4 °C overnight in Can Get Signal @ Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan) against total HSL (Abcam, Cambridge, UK), HSL phosphorylated at Ser-563 (Cell Signaling Technology [CST], Danvers, MA, USA), HSL phosphorylated at Ser-660 (CST), ATGL (CST), total perilipin 1 (Abcam), CGI-58 (Santa Cruz Biotechnology, Dallas, TX, USA), PPARγ2 (ThermoFisher Scientific, Waltham, MA, USA), BMAL1 (Abcam), AKAP150 (Alomone Labs, Jerusalem, Israel), PKARIα (BD biosciences, Franklin Lakes, NJ, USA), PKARIIα (Abcam), PKARIIβ (Abcam), and β-actin (Abcam).

Techniques: Isolation, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Exercise Training-Enhanced Lipolytic Potency to Catecholamine Depends on the Time of the Day

doi: 10.3390/ijms21186920

Figure Lengend Snippet: Association of BMAL1 with several lipolytic machineries in primary adipocytes isolated from epididymal adipose tissues. ( A ) Direct interaction between BMAL1 and BMAL1 revealed by the co-immunoprecipitation assay. Both lysates and immunoprecipitates (IP) were subjected to immunoblotting with ( B ) anti-HSL, ( C ) anti-ATGL, ( D ) anti-Perilipin1, ( E ) anti-AKAP150, ( F ) anti-PKA-RIα, ( G ) anti-PKA-RIIα, and ( H ) anti-PKA-RIIβ antibodies. Relative protein expression levels of each band in E-EX and L-EX groups (levels in respective sedentary controls are set to 1). In all experiments, data are presented as the means ± S.E. ( n = 6). * p < 0.05. The abbreviations for the groups of rats are shown in and .

Article Snippet: After separation by 6–12.5% SDS-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (ATTO), blocked for 60 min with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim milk, or with Block Ace ® Powder (DSP, Osaka, Japan) dissolved in purified water, and were then probed at 4 °C overnight in Can Get Signal @ Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan) against total HSL (Abcam, Cambridge, UK), HSL phosphorylated at Ser-563 (Cell Signaling Technology [CST], Danvers, MA, USA), HSL phosphorylated at Ser-660 (CST), ATGL (CST), total perilipin 1 (Abcam), CGI-58 (Santa Cruz Biotechnology, Dallas, TX, USA), PPARγ2 (ThermoFisher Scientific, Waltham, MA, USA), BMAL1 (Abcam), AKAP150 (Alomone Labs, Jerusalem, Israel), PKARIα (BD biosciences, Franklin Lakes, NJ, USA), PKARIIα (Abcam), PKARIIβ (Abcam), and β-actin (Abcam).

Techniques: Isolation, Co-Immunoprecipitation Assay, Western Blot, Expressing