AMPK Methods and Protocols

3.4 Harvesting
of the Cells

1. After stimulation and/or transfection (see Subheadings
   3.1–3.3), remove the coverslips from the dishes using a forceps
   and transfer into a 12-well plate containing PBS (seeNote 25).
   The continued procedure of immunostaining is described in
   Subheading3.6.

A

C

3 T

3 H

IN

SO

2U

D

B

ULK1

135 P-ULK1 (Ser555)

135

• -GluA+A

NIH3T3

AMPK

P-AMPK (Thr172)

LC3-I

p62

actin

LC3-II

63

63

63

48

17

ACC

245 P-ACC (Ser79)

245

Raptor

(^180) P-Raptor (Ser792)
180
LC3-II/actin: 00.1 23 31. 4.4
P-ULK1/ULK1: 00.1 36 05. 6.2
ULK1
135 P-ULK1 (Ser555)
135
-GluA+A
U2OS
AMPK
P-AMPK (Thr172)
LC3-I
p62
actin
LC3-II
63
63
63
48
17
ACC
245 P-ACC (Ser79)
245
Raptor
(^180) P-Raptor (Ser792)
180
LC3-II/actin: 00.1 16 18. 7.2
P-ULK1/ULK1: 00.1 14 8. 8 2.2

• -GluA+A
  
  
  • -GluA+A
  
  
  
  

0

10

20

30

40

50

LC3

dots per

cell

NIH3T3

U2OS

***

****

**

***

• 
  


• -Glu A+A


• -Glu A+A

Fig. 1Evaluation of the induction of AMPK-mediated autophagy by immunoblotting and immunofluorescence.
(a) NIH3T3 and (b) U2OS cells were cultured in glucose-free medium (-Glu) or treated with AICAR and
A-769662 (AþA) as indicated and subjected to immunoblot analysis using the indicated pan- and phospho-
antibodies. In the two cell lines, both treatments result in an activation of AMPK seen by increased
phosphorylation of the AMPK substrates ACC, ULK1, and Raptor and the induction of autophagy displayed
by a modest increase in the amount of LC3-II. (c) Representative confocal images of NIH3T3 and U2OS cells
treated as described in (a) and (b). Staining of endogenous LC3 was used to monitor autophagy induction
measured as an increase in punctate LC3. Scale bar, 20μm. (d) Quantification of the counted LC3 dots per cell
of the cells shown in (c) for each treatment (n>100). Data are shown as mean valuesSD. The significance
was tested by applying a Student’st-test (**p0.05, *p0.01, **p0.001)

380 Sarah Krieg et al.