Rabbit anti hsl

The following primary antibodies were used for Western blotting: rabbit-anti-Insulin receptor β (Santa Cruz, #711), rabbit-anti-phospho Akt (Ser473) (Cell Signaling, #4060), rabbit-anti-Akt (Cell Signaling, #9272), rabbit-anti Gsk3β (Cell Signaling, #9315), rabbit-anti-phospho Gsk3β Ser9 (Cell Signaling, #5558), rabbit-anti FoxO1 (Cell signaling, #2880), rabbit-anti-phospho FoxO1 Ser256 (Cell Signaling, #9461), rabbit-anti p70 S6 kinase (Cell Signaling, #2708), mouse-anti-phospho p70 S6 kinase Thr389 (Cell Signaling, #9206), mouse-anti S6 (Cell Signaling, #2317), rabbit-anti-phospho S6 Ser235/236 (Cell signaling, #4856), rabbit-anti 4E-BP1 (Cell Signaling, #9644), rabbit-anti-phospho 4E-BP Thr37/46 (Cell Signaling, #9459), mouse-anti-Gapdh (Abcam #ab8245), rabbit-anti-phospho HSL Ser660 (Cell Signaling, #4126), rabbit-anti-phospho HSL Ser563 (Cell Signaling, #4139), rabbit-anti-HSL (Cell Signaling, #4107), rabbit-anti-PLIN1 (Cell Signaling, #9349) and rabbit-anti-CD36 (Cell Signaling #14347). Secondary antibodies used for western blotting were goat-anti-rabbit IgG-HRP conjugate (Biorad, #1706515) and goat-anti-mouse IgG-HRP conjugate (Biorad, #1706516).

Proteins of cell lysates or tissue homogenates were separated by SDS–PAGE according to their molecular weight using Tris/glycine as electrophoresis buffer and were transferred onto polyvinylidene fluoride membranes (Carl Roth GmbH, Karlsruhe, Germany) using CAPS buffer. After blocking, membranes were hybridized with respective primary antibodies. Membranes were washed, incubated with respective secondary horseradish-peroxidase (HRP)-conjugated antibody and detected using ECL2 Western blotting substrate (Thermo Scientific, Waltham, MA). Antibodies used were rabbit anti-HSL, rabbit anti-phospho-HSL (S660), and rabbit anti-GAPDH (all from Cell Signaling, Danvers, MA, USA), rabbit anti-CGI-58 (Abnova, Heidelberg), mouse anti-β-Actin (Santa Cruz, Santa Cruz, CA, USA), rabbit anti-alpha smooth muscle actin (α-SMA, Pierce, Thermo Scientific), HRP-linked sheep-anti mouse antibody (GE Healthcare Amersham, Buckinghamshire, UK), and HRP-linked rat-anti rabbit antibody (Dako, Glostrup, Denmark).

The cells were homogenized in RIPA lysis buffer containing protease inhibitors. The protein concentrations were measured using the BCA method. Proteins (100 mg) were separated on 10% SDS-PAGE gels and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked in 5% (w/v) non-fat milk for 1 h and then incubated with rabbit anti-p-HSL (Cell Signaling, Beverly, MA, USA; 1:1000 dilution), rabbit anti-HSL (Cell Signaling, Beverly, MA, USA; 1:1000 dilution), rabbit anti-ATGL (Cell Signaling, Beverly, MA, USA; 1:1000 dilution), rabbit anti-p-AMPK (Thr 172), rabbit anti-AMPK (Cell Signaling, Beverly, MA, USA; 1:1000 dilution), rabbit anti-PPARγ (Millipore, USA; 1:1000 dilution) or mouse anti-β-actin (Proteintech, Chicago, IL, USA; 1:2000 dilution) primary antibodies overnight at 4 °C. The membranes were then incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h at room temperature. After washing with TBST, the immune complexes were detected using the Alpha Q Chemiluminescence System and exposed to film. The relative intensity of the target protein to HSL or to β-actin in the same sample was analyzed using the Alpha Q software.