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Rabbit anti p ampk t172

Manufactured by Cell Signaling Technology
Sourced in United States

Rabbit anti-p-AMPK (T172) is a primary antibody that specifically recognizes the phosphorylated form of AMP-activated protein kinase (AMPK) at threonine 172. AMPK is a cellular energy sensor that plays a crucial role in regulating cellular metabolism and energy homeostasis.

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4 protocols using rabbit anti p ampk t172

1

Western Blot Analysis of Cell Signaling Proteins

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Cells in microslides were washed twice with ice-cold PBS and lysed on ice with 150 μl of 1X Laemmli buffer (60 mM Tris-HCl pH=6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95°C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (Sigma, Cat#L7543); rabbit-anti-FLCN (Cell signaling, Cat#3697); rabbit-anti-FNIP1 (Abcam, Cat#ab134969); rabbit-anti-AMPK (Cell signaling, Cat#2532S); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); mouse-anti-actin (Millipore, Cat#1501); rabbit-anti-ATG16L1 (MBL, Cat#PM040); rabbit-anti-IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti β-catenin (Cell signaling, Cat#8480); rabbit-anti-LKB1 (Cell signaling, Cat#3050); rabbit-anti-S6 ribosomal protein (Cell signaling, Cat#2217); rabbit-anti-p-S6 ribosomal protein (S240/244) (Cell signaling, Cat#2215); rabbit-anti-Tuberin/TSC2 (Cell signaling, Cat#4308); rabbit-anti-p-Tuberin/TSC2 (T1462) (Cell signaling, Cat#3617). Secondary HRP conjugate anti-rabbit IgG (GE Healthcare) and HRP conjugate anti-mouse IgG (Bio-Rad).
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2

Adiponectin Receptor and AMPK Signaling

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Cell homogenates (20 μg/well) were loaded onto 10% SDS polyacrylamide gels in denaturing conditions at 80 mA for 90 min and transferred electrophoretically (100 mA/blot, 2 h; Power Pack; Bio-Rad Laboratories, Inc., USA) to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed as described previously [48 (link)]. Nonspecific binding was blocked with 5% non-fat milk powder in Tris-buffered saline-Tween containing 0.1% Tween-20 (PBS-T) for 1 h. Primary antibodies including rabbit anti-AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-AdipoR2 (1:1000, Boster Biological Technology, USA), rabbit anti-AMPK (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-AMPKT172 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-α-Tubluin (1:5000, Cell Signaling Tech. Inc., USA), rabbit anti-p-NF-κB p65S536 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-IκBα (Ser32) (1:1000, Cell Signaling Tech. Inc., USA), mouse anti-IκBα (1:1000, Cell Signaling Tech. Inc., USA) antibody were incubated at 4 °C overnight, followed by HRP-conjugated secondary antibodies (goat anti-rabbit, 1:5000 or rabbit anti-mouse, 1:5000; Dako, Glostrup, Denmark) at RT for 1 h. The immunoblot signals were visualized by Westernbright Quantum HRP substrate (advansta, USA).
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3

Immunofluorescent Microscopy of Axoneme Proteins

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Cells were fixed either with 4% paraformaldehyde (PFA) for 20 min or with cold methanol for 5 min at -20°C for proper axoneme proteins detection [22 (link)]. They were then washed and incubated for 30 min in blocking buffer (10% FCS in PBS) followed by incubation with primary antibodies diluted in blocking buffer supplemented with 0.05% saponin for 1 h at room temperature or overnight at 4°C. Cells were washed 3 times, and then incubated for 1 h with fluorescent Alexa-Fluor secondary antibodies. After washing, 150 μl of DAPI-Fluoromount were added into the Luer chamber (Southern Biotech). Images were acquired with a Zeiss Apotome.2 fluorescence microscope equipped with a 63x oil immersion fluorescence objective. Number of ciliated cells and length of cilia were quantified using Zen Software (Zeiss) or Imaris Software (Bitplane). The following antibodies were used for immunofluo-rescence: mouse-anti-LC3B (MBL, Cat# M152-3); rabbit-anti-FLCN (Cell signaling, Cat#3697); mouse-anti-ARL13B (C-5) (Santa Cruz, Cat#515784); rabbit-anti-ATG16 (MBL, Cat#PM040); rabbit-anti IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); Phalloidin (Cat# A34055); Alexa Fluor-conjugated secondary anti-bodies (donkey anti-mouse IgG and donkey anti-Rabbit IgG, Life Technologies).
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4

Immunofluorescence Analysis of Drosophila Eye Discs

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Third instar larval eye-imaginal discs were dissected in 1× phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min on ice. Discs were then washed three times for 10 min each in ice-cold 1× PBS, permeabilized in 0.3% Triton X100/1× PBS (PBST) for 20 min at RT, and washed again three times for 10 min each before blocking in 10% normal goat serum in 0.1% PBST for 30 min at RT. Discs were incubated in primary antibodies (4 °C overnight) in 10% normal goat serum/0.1% PBST. The following day, discs were washed three times for 5 min each in 0.1% PBST before incubating in secondary antibodies (in the dark at RT for 1 h) in 10% NGS/0.1% PBST. Finally, discs were washed three times for 10 min each in 1× PBS at RT and mounted using VectaShield anti-fade mounting medium. Primary antibodies and dilution: rabbit anti-cleaved Drosophila DCP1 (Asp216) (Cell Signaling, 1:100), mouse anti-MMP1 (3A6B4/5H7B11/3B8D12 antibodies were mixed in equal amounts) (DSHB, 0.2 µg/ml), and Rabbit anti-pAMPK (T172) (Cell Signaling 1:100). Fluorescent secondary antibodies were from Life Technologies. DAPI was used to stain DNA.
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