AMPK Methods and Protocols

3.4 Harvesting of the Cells After stimulation and/or transfection (see Subheadings 3.1–3.3), remove the coverslips from the dis ...

B C A -Glu Baf. A1 -Glu Baf. A1 -Glu Baf. A1 -Glu Baf. A1 siControl siAMPK ULK1 135 P-ULK1 (Ser555) 135 siControl − −+ − − ...

Thereafter wash the cells attached to the dish with ice-cold PBS and lyse in 400μl (10 cm dish) or 150μl (6 cm dish) of Frackel ...

Dilute 30μg of total protein with lysis buffer to 15μl of total volume, mixed with 5μlof4loading buffer, and heat at 95 C for ...

Thereafter wash the blots with TBS-T three times for at least 5 min each on a horizontal shaker. Afterward incubate the blot wi ...

Thereupon wash the coverslips two times with PBS and one time with H 2 O(seeNote 9). Add 500μl of Hoechst solution for 5 min fo ...

In our experience, the antibodies listed in Subheading2.5 worked well, which is why we share the supplier information. However, ...

than two coverslips should be mounted in parallel since the Mowiol spots would dry out otherwise. Cells need to be tested for m ...

The transfection immediately after seeding the cells saves 1 day and increases the transfection efficiency. The time necessary ...

To keep the box humid, a wet paper towel was placed in the closed box together with the coverslips on top of the Parafilm. For ...

AICAR and A769662. Am J Phys Endocrinol Metab 306(6):E688–E696.https://doi.org/ 10.1152/ajpendo.00672.2013 Hardie DG, Ross FA, ...

Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a ...

Chapter 25 Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis Chen-Song Zhang, Mengqi Li, Yue Zong, ...

requires concerted action of AXIN, the lysosomal v-ATPase, and the Ragulator complex (a pentamer consisting of LAMTORs 1-5) [15, ...

The adenine nucleotides AMP, ADP, and ATP exert various effects on AMPK activity [11, 12]. Moreover, the cellular energy levels ...

2 Materials 2.1 Analysis of AMPK Activation in Cells or Mice Glucose-free DMEM: glucose-free Dulbecco’s Modified Eagle’s Medium ...

1160 mm centrifuge tubes. Buffer A: 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mMβ-mercaptoethanol, 0.25% (v/v) Triton ...

Okadaic acid: 10 μg/mL okadaic acid in DMSO. Store at
20 C. Recombinant His-AXIN (full length), expressed inE.coli. Recombin ...

Rinse cells twice with 2 mL of PBS (RT). Add 2 mL of complete DMEM medium or glucose-free DMEM supplemented with desired concen ...

3.2 Analysis of the Lysosomal Localization of AXIN/ LKB1 Preparation of Lysosomes In this section, the method preparation of lys ...