Anti-AMPK gamma 1 antibody [Y308]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling AMPK gamma 1 with ab32508 at 1/100 dilution (1μg) (Red). Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were fixed with4% paraformaldehyde. Rabbit monoclonal IgG (ab172730) was used as isotype control (Black). Unlabelled control : Cells without incubation with primary antibody and secondary antibody (Blue).

• IP

Lab

Immunoprecipitation - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

AMPK gamma 1 was immunoprecipitated using 1 mg Jurkat whole cell extract, 0.2 ug of Rabbit monoclonal [Y308] to AMPK gamma 1and 50μl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10min under agitation. No antibody was added to the control (lane 2). Jurkat whole cell extractdiluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70^oC; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab32508. Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Bands : 37kDa : AMPK gamma 1.

All lanes:

Immunoprecipitation - Anti-AMPK gamma 1 antibody [Y308] (ab32508)

Predicted band size: 38 kDa

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• WB

AbReview39625****

Western blot - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

All lanes:

Western blot - Anti-AMPK gamma 1 antibody [Y308] (ab32508) at 1/1000 dilution

All lanes:

HEK293 whole cell lysate at 30 µg

Secondary

All lanes:

Alexa Fluor® 690-conjugated Goat anti-rabbit IgG polyclonal at 1/10000 dilution

Predicted band size: 38 kDa

Observed band size: 38 kDa

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Exposure time: 5min

This image is courtesy of an anonymous Abreview

• WB

Unknown

Western blot - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

All lanes:

Western blot - Anti-AMPK gamma 1 antibody [Y308] (ab32508) at 1/1000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 38 kDa

Observed band size: 38 kDa

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• WB

CiteAb

Western blot - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

Western Blotting using Anti-AMPK gamma 1 antibody [Y308], ab32508. Publication image from Yan, Z. et al., 2017, Nat Commun, 28916822. Legend direct from paper.

Ampk, Ulk1 and Drp1, are transiently activated immediately following acute exercise. Ampk and its substrate Acc are phosphorylated at Thr172 a, b and S79 a, c, respectively, immediately after the cessation of exercise and return to baseline values by 3 h post-exercise in C57BL/6 J mice aged 10–12 weeks. Ulk1 is phosphorylated at Ser555, but not Ser467 or Ser757, immediately following acute exercise, which returns to basal levels by 3 h post-exercise a, d–f. Drp1 is phosphorylated in skeletal muscle at Ser616 a, g immediately after the cessation of exercise and Ser637 a, h reaching significance 3 and 6 h after exercise. Mfn2 protein abundance is unchanged following acute treadmill running a, i. Representative western blots are shown a and all quantification is presented as protein phosphorylation to total protein abundance ratio b–f. Data presented as mean ± standard error of the mean. n = 5 (except 24 h, n = 4). See also Supplementary Figs. 6 and 7. Results of one-way ANOVAs are *p < 0.05, **p < 0.01. F = 6.44 for b, F = 3.56 for c, F = 6.50 for d, F = 0.89 for e, F = 0.33 for f, F = 3.34 for g, F = 2.76 for h, F = 0.31 i. DF = 5. p-Ampk (T172/Total) b was sqrt transformed to account for unequal variance., DF = 5. S637/total Drp1 data was log transformed to account for unequal variance

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• WB

CiteAb

Western blot - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

Western Blotting using Anti-AMPK gamma 1 antibody [Y308], ab32508. Publication image from Yan, Z. et al., 2017, Nat Commun, 28916822. Legend direct from paper.

Ampk is necessary and sufficient for phosphorylation of Ulk1 at Ser555 in response to acute exercise. Phosphorylation of Ampk at T172 in response to acute exercise is blocked in Ampk dnTG a, b, whereas increased phosphorylation of Acc at Ser79 post-exercise is still observed a, c. In Ampk caTG mice, both T172 Ampk and S79 Acc are elevated in sedentary conditions f–h. Phosphorylation of Ulk1 at Ser555 is blunted in dnTG skeletal muscle immediately after acute exercise a, d but elevated in caTG mice basally f, i. There was no effect on S757 phosphorylation in either dnTG mice post exercise a, e or sedentary caTG mice (f, j). Representative western blots are shown for dnTG mice a and caTG mice f. See also Supplementary Figs. 8 and 9. All quantification is presented as protein phosphorylation to total protein abundance ratio b–e, g–j. Data are presented as mean ± standard error of the mean. For dnTG study n = 10 WT or 5–7 dnTG (12–13 weeks). Results of two-way ANOVAs are *p < 0.05, **p < 0.01 for sedentary vs. exercise comparisons, F = 4.57 and DF = 1 c. ###p < 0.001 for between group comparisons, F = 14.03 and DF = 1 (e). Tukey multiple comparison tests were performed when a significant interaction effect was observed, in which *p < 0.05, **p < 0.01 for WT sedentary vs. WT exercise comparisons and $$p < 0.01, $$$p < 0.001 for WT exercise vs. dnTG exercise comparisons, DF = 27. For caTG study n = 3 (WT) or 5 (caTG) (13–15 weeks). Results of two-tailed t-tests are *p < 0.05 and **p < 0.01 for WT to caTG comparisons, t = 4.94 (g), t = 2.57 (h), t = 2.71 (i), t = 1.72 (j), DF = 6

false

• WB

CiteAb

Western blot - Anti-AMPK gamma 1 antibody [Y308] (AB32508)

Western Blotting using Anti-AMPK gamma 1 antibody [Y308], ab32508. Publication image from Yan, Z. et al., 2017, Nat Commun, 28916822. Legend direct from paper.

Ampk is necessary and sufficient for phosphorylation of Ulk1 at Ser555 in response to acute exercise. Phosphorylation of Ampk at T172 in response to acute exercise is blocked in Ampk dnTG a, b, whereas increased phosphorylation of Acc at Ser79 post-exercise is still observed a, c. In Ampk caTG mice, both T172 Ampk and S79 Acc are elevated in sedentary conditions f–h. Phosphorylation of Ulk1 at Ser555 is blunted in dnTG skeletal muscle immediately after acute exercise a, d but elevated in caTG mice basally f, i. There was no effect on S757 phosphorylation in either dnTG mice post exercise a, e or sedentary caTG mice (f, j). Representative western blots are shown for dnTG mice a and caTG mice f. See also Supplementary Figs. 8 and 9. All quantification is presented as protein phosphorylation to total protein abundance ratio b–e, g–j. Data are presented as mean ± standard error of the mean. For dnTG study n = 10 WT or 5–7 dnTG (12–13 weeks). Results of two-way ANOVAs are *p < 0.05, **p < 0.01 for sedentary vs. exercise comparisons, F = 4.57 and DF = 1 c. ###p < 0.001 for between group comparisons, F = 14.03 and DF = 1 (e). Tukey multiple comparison tests were performed when a significant interaction effect was observed, in which *p < 0.05, **p < 0.01 for WT sedentary vs. WT exercise comparisons and $$p < 0.01, $$$p < 0.001 for WT exercise vs. dnTG exercise comparisons, DF = 27. For caTG study n = 3 (WT) or 5 (caTG) (13–15 weeks). Results of two-tailed t-tests are *p < 0.05 and **p < 0.01 for WT to caTG comparisons, t = 4.94 (g), t = 2.57 (h), t = 2.71 (i), t = 1.72 (j), DF = 6

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