Phospho-CaMKK2 (Ser511) Antibody - #AF4487

Fig. 2. NCAPD2 inhibited cell autophagy and disrupted autophagic flux via Ca 2+ /CAMKK2/AMPK/mTORC1 pathway. (A) Western blot analyses for phosphorylated mTOR (p-mTOR, S2448), phosphorylated p70S6K (p-p70S6K, T389/412), phosphorylated 4E-BP1 (p-4E-BP1, T70) and phosphorylated AKT (p-AKT, S473) in CRCC cells with different treatments as indicated. (B) Western blot of indicated proteins in cells treated with mTORC1 inhibitor Rapamycin (3 nM, 24h). (C) Immuno- fluorescence staining of LC3II (red) and P62 (red) in CRC cells with different treatments as indicated. Merged images represented overlays of LC3II or P62 and nuclear staining by DAPI (blue). (D) Intracellular Ca 2+ levels were analyzed by flow cytometry after staining with the fluorescent probe Fluo-3, AM in CRC cells. (E) Representative Western blot gel documents of phosphorylated CAMKK2(S511), phosphorylated AMPK(T172), phosphorylated mTOR(S2448), Beclin, ATG5, P62, LC3II in CRC cells with different treatments. (F) Western blots of indicated proteins in cells treated with an inhibitor of microsomal Ca 2+ -ATPase Thapsigargin (1 μ M, 6h) and Ca 2+ chelator BAPTA-AM (10 μ M, 12h) respectively. Results are shown as mean ± s.d, *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7 L-theanine regulates hepatocyte lipid metabolic pathways via the CaMKKβ-AMPK signaling pathway. HepG2 cells were treated with 500 μM OA for 24 h with or without pretreated L-theanine for 2 h. A Western blot analysis showing protein expression of p-CaMKKβ and p-AMPK in HepG2 cells. HepG2 cells were pretreated with 10 μM STO-609 for 2 h, then added L-theanine co-incubated for 2 h, finally, OA were added co-incubated for 24 h. B Abrogating effect of CaMKKβ inhibitor on L-theanine-facilitated phosphorylation of CaMKKβ and AMPKα in HepG2 hepatocytes. HepG2 cells were pretreated with 10 μM BAPTA-AM for 1 h, then added L-theanine co-incubated for 2 h, finally, OA were added co-incubated for 24 h. C Intracellular Ca2+ levels were assessed in HepG2 cells with Fluo-4 AM using a fluorescence microscope. Scale bar: 20 μm (20 ×). D [Ca2+]i was detected by a multifunctional microplate reader using Fluo-4 AM. under the condition of 488 nm excitation wavelength and 520 nm emission wavelength. E Western blots of indicated proteins in HepG2 cells treated with an intracellular Ca2+ chelator BAPTA-AM. Band intensity was quantified by densitometry analysis. Values are expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, versus control group; #p < 0.05, ##p < 0.01, versus OA group; &p < 0.05 versus OA + STO-609 group, $p < 0.05 versus OA + BAPTA-AM group. n.s.: not significant (p > 0.05) versus control group

Figure 2: |Effect of CADE on miR-378b-CaMKK2 signal. (a) The expression level of miR-378b in liver tissues. (b) CaMKK2 mRNA expression. (c) Protein expression levels of CaMKK2 and p-CaMKK2. All data are expressed as means ± SD.

Figure 5 Western blot analysis of AMPK, CAMKKβ, AMPK phosphorylation at Thr-172 and CAMKKβ phosphorylation at Ser-511 in the homogenates of myocardial tissue from rat hearts. (A) Western blot bands of AMPK and p-AMPK(Thr172) protein and the ratio of p-AMPK(Thr172)/total AMPK. (B) Western blot bands of CAMKKβ and p-CAMKKβ(Ser511) protein and the ratio of p-CAMKKβ(Ser511)/total CAMKKβ. APC-induced increases of AMPK and CAMKKβ phosphorylation were reduced by the muscarinic acetylcholine receptor antagonist atropine (ATR, 100 nM) and nicotinic acetylcholine receptor antagonist hexamethonium (HEM, 50 µM). The data are presented as the mean ± standard deviation. n=5 hearts/group. *P<0.05 vs. Sham group; &P<0.05 vs. IR group; #P<0.05 vs. APC group. CAMKKβ, calcium/calmodulin-dependent protein kinase kinase β; p, phosphorylated; IR, ischemia-reperfusion; APC, anesthetic preconditioning; ATR, atropine; HEM, hexamethonium; CTL, control.