phospho ampk alpha pab antibodies  (Proteintech)

Journal: Plant Physiology

Article Title: DOMAIN OF UNKNOWN FUNCTION581-9 negatively regulates SnRK1 kinase activity

doi: 10.1093/plphys/kiad594

Figure Lengend Snippet: DUF581-9 interferes with KIN10 T175 T-loop phosphorylation. A) Western blot analysis of recombinant MBP-KIN10 (full length) kinase assay using an anti-MBP and anti-phospho-α-AMPK (T172) antibody specifically detecting phosphorylated KIN10 T175. Densitometric analysis mirrors the quantification of KIN10 T-loop phosphorylation. B) Mapping of KIN10 phosphorylation sites by LC-MS/MS. Schematic representation of robustly identified phosphorylation sites. Table indicates the relative changes in phosphorylation, calculated for different phosphorylation sites within the KIN10 polypeptide and compared with a positive control without DUF581-9 or DUF581-9 C47S . Three technical replicates were measured and used for calculation. All experiments were carried out 3 times with similar results. Asterisks (* P < 0.05, *** P < 0.001, **** P < 0.0001) mark significant differences according to 1-way ANOVA analysis followed by Tukey’s multiple comparison test.

Article Snippet: To detect KIN10 T-loop phosphorylation, a phospho-AMPK alpha-1 (Thr172) polyclonal antibody (Cell Signalling Technology) was used (1:500).

Techniques: Phospho-proteomics, Western Blot, Recombinant, Kinase Assay, Liquid Chromatography with Mass Spectroscopy, Positive Control, Comparison

Journal: Plant Physiology

Article Title: DOMAIN OF UNKNOWN FUNCTION581-9 negatively regulates SnRK1 kinase activity

doi: 10.1093/plphys/kiad594

Figure Lengend Snippet: DUF581-9 interferes with KIN10/GRIK2 interaction in vivo and in vitro leading to diminished KIN10 T175 T-loop phosphorylation in vivo. A) Validation of KIN10 and GRIK2 interaction after KIN10 myc:IP (immunoprecipitation). Furthermore, KIN10 T-loop phosphorylation is weakened by DUF581-9 but not by KIN10-binding inefficient DUF581-9 C47S protein. Proteins were transiently expressed in leaves of N. benthamiana using Agrobacterium infiltration. Samples were harvested 48 h post infiltration. Crude extract mirrors input signal of KIN10 (anti-myc), GRIK2 or KINß2 (anti-HA), and DUF581-9/DUF581-9 C47S (anti-mCherry) protein. For KIN10 T-loop phosphorylation, immunoblot analysis was performed with anti-P T172 AMPK antibody. Densitometric analysis of anti-P T172 AMPK signal confirms the weaker KIN10 T-loop phosphorylation only in the presence of DUF581-9 protein. Pulldown of KIN10 protein was performed by anti-myc affinity matrix followed by immunoblot analysis using anti-myc, anti-HA, or anti-mCherry antibodies. Densitometric analysis of KIN10 Venus N173 and GRIK2-Venus C155 association confirms the weaker protein interaction of KIN10 and GRIK2 in the presence of DUF581-9. The homomerization of KIN10-Venus N173 and KINß2-Venus C155 was used as control and is not affected by DUF581-9 mCherry and DUF581-9 C47S mCherry. B) Proteins were mixed as indicated, and KIN10-GST (full length) was pulled down from the mixture. Recombinant proteins were detected before (Input) and after (IP:GST) by immunoblotting using anti-MBP or anti-GST antibodies. Red box highlights GRIK2 MBP protein abundance after KIN10 IP:GST. The experiment was carried out at least twice with similar results. Asterisks indicate an unspecific protein band present in MBP empty vector control.

Article Snippet: To detect KIN10 T-loop phosphorylation, a phospho-AMPK alpha-1 (Thr172) polyclonal antibody (Cell Signalling Technology) was used (1:500).

Techniques: In Vivo, In Vitro, Phospho-proteomics, Biomarker Discovery, Immunoprecipitation, Binding Assay, Western Blot, Control, Recombinant, Quantitative Proteomics, Plasmid Preparation

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Curcumin protects human umbilical vein endothelial cells against high oxidized low density lipoprotein-induced lipotoxicity and modulates autophagy

doi: 10.22038/IJBMS.2021.59969.13297

Figure Lengend Snippet: Curcumin ameliorated autophagy inhibition induced by high ox-LDL in HUVECs through the AMPK/mTOR/p70S6K pathway. A and B: The protein expression levels of LC3-II were measured by Western blotting (n=3). C and D: The protein expression levels of AMPK and p-AMPK were measured by Western blotting (n=3). E and F: The protein expression levels of mTOR and p-mTOR were measured by Western blotting (n=3). G and H: The protein expression levels of p70S6K and p-p70S6K were measured by Western blotting. Expression of these proteins was quantified by densitometry using ImageJ software (n=3). The data are shown as the means±SDs (* P < 0.01 vs. the control group; ** P < 0.01 vs. the control group; # P <0.05 vs. the high ox-LDL group; ## P < 0.01 vs. the high ox-LDL group; & P < 0.05 vs. the compound C group). AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 protein kinase

Article Snippet: Protein extracts were isolated, separated on a 12.5% SDS-PAGE gel and transferred onto a 0.22 μm PVDF membrane (Epizyme), which was incubated with the following primary antibodies: peroxisome proliferator-activated receptor γ (PPARγ) (16001-1-AP, Proteintech, Wuhan, China), interleukin-6 (IL-6) (ab233706, Abcam, Cambridge, 1:1000), interleukin-10 (IL-10) (ab133575, Abcam, 1:5000), tumor necrosis factor alpha (TNF-α) (17590-1-AP, Proteintech, Wuhan, China), microtubule-associated protein 1 light chain 3 (LC3) (2775S, Cell Signaling Technology, MA, USA, 1:1000), AMPK (HN0506, HuaBio, Hangzhou, China, 1:1000), mTOR (HN0824, HuaBio, 1:500), p70S6K (HJ0505, HuaBio, 1:1000), phosphorylated AMPK (p-AMPK) (bs-8813R, Bioss, Beijing, China, 1:1000), phosphorylated mTOR (p-mTOR) (2971S, Cell Signaling Technology, 1:500), and phosphorylated p70S6K (p-p70S6K) (9234S, Cell Signaling Technology, 1:1000). β-Actin (AC026, ABclonal, Wuhan, China, 1:50000) was used as an internal control.

Techniques: Inhibition, Expressing, Western Blot, Software