Total proteins were extracted by RIPA. Proteins were separated by SDS-PAGE and electro-transferred onto PVDF membranes. The membranes were soaked in buffer containing 5% BSA for 1 h and then incubated with primary antibodies including anti‐p-mTOR (5536s, 1:1000, Cell Signaling Technology), anti‐mTOR (2983s, 1:1000, Cell Signaling Technology), anti‐p-PI3K (bs-6417R, 1:1000, Bioss), anti‐PI3K (bs-10657R, 1:1000, Bioss), anti‐p-AMPK (2531S, 1:1000, Cell Signaling Technology), anti‐AMPK (5832S, 1:1000, Cell Signaling Technology), anti‐p-AKT (Sc-293125, 1:1000, Santa), anti‐AKT (Sc-5298, 1:1000, Santa), anti‐PGC1α (bs-7535R, 1:1000, Bioss), anti‐LC3B (ab51520, 1:5000, Abcam), anti‐ZO-1 (61–7300, 1:1000, Invitrogen), anti‐Occludin (71–1500, 1:1000, Invitrogen), or anti‐Claudin-5 (34–1600, 1:1000, Invitrogen), at 4 °C overnight. Thereafter, the membranes were washed with Tris‐buffered saline Tween buffer and incubated with HRP‐rabbit (ab191866, 1:5000, Abcam) or HRP‐mouse (bs-0296G-HRP, 1:1000, Bioss) conjugated secondary antibody for 1 h at room temperature (RT). An Odyssey Infrared Imaging System was used to scan the membranes for further analysis. β-actin (CL594-66009, 1:1000, Proteintech) was used as loading control. Band intensities were quantified using Image J software.
Anti p mtor 5536s
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti‐p-mTOR (5536s) is a lab equipment product manufactured by Cell Signaling Technology. It is an antibody that specifically binds to the phosphorylated form of the mammalian target of rapamycin (mTOR) protein. The core function of this product is to detect and measure the activated state of the mTOR signaling pathway in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab51520, by Abcam (1 mentions)
Ab191866, by Abcam (1 mentions)
Anti mtor 2983s, by Cell Signaling Technology (1 mentions)
Anti pi3k bs 10657r, by Bioss Antibodies (1 mentions)
Anti ampk 5832s, by Cell Signaling Technology (1 mentions)
Bs 0296g hrp, by Bioss Antibodies (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti claudin 5, by Thermo Fisher Scientific (1 mentions)
β actin, by Proteintech (1 mentions)
2 protocols using anti p mtor 5536s
1
Protein Expression Analysis by WB
2
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total cellular protein was extracted
and detected by the BCA method; the proteins were denatured and separated
by 10% SDS polyacrylamide gel electrophoresis and transferred to a
polyvinylidene fluoride (PVDF) membrane by the semi-dry transfer method,
5% off Lipid milk powder closed 2 h, anti-P62 (1:1000), anti-LC3 (1:1000),
anti-Beclin1 (1:1000), anti-AMPK (1:1000), anti-p-AMPK (1:1000), anti-DDiT4 (1:1000), anti-mTOR (1:1000), and anti-p-mTOR (1:1000) were added at 4 °C overnight, secondary
antibody (1: 3000) was incubated for 1 h at room temperature, and
the optical density of the bands was measured by ImageJ software.
Anti- P62 (GB11239-1) and anti-LC3 (GB11124) were obtained from Servicebio
(Wuhan, Hubei, China). Anti-Beclin1 (PD017) was provided from MBL
(Nagoya, Aichi, Japan). Anti-AMPK (2532S), anti-p-AMPK (2535S), anti-mTOR (2972S), and anti-p-mTOR
(5536S) were obtained from Cell Signaling Technology (Beverly, MA,
USA). Anti-DDiT4 (ab106356) was purchased from Abcam (Cambridge, MA,
USA).
and detected by the BCA method; the proteins were denatured and separated
by 10% SDS polyacrylamide gel electrophoresis and transferred to a
polyvinylidene fluoride (PVDF) membrane by the semi-dry transfer method,
5% off Lipid milk powder closed 2 h, anti-P62 (1:1000), anti-LC3 (1:1000),
anti-Beclin1 (1:1000), anti-AMPK (1:1000), anti-p-AMPK (1:1000), anti-DDiT4 (1:1000), anti-mTOR (1:1000), and anti-p-mTOR (1:1000) were added at 4 °C overnight, secondary
antibody (1: 3000) was incubated for 1 h at room temperature, and
the optical density of the bands was measured by ImageJ software.
Anti- P62 (GB11239-1) and anti-LC3 (GB11124) were obtained from Servicebio
(Wuhan, Hubei, China). Anti-Beclin1 (PD017) was provided from MBL
(Nagoya, Aichi, Japan). Anti-AMPK (2532S), anti-p-AMPK (2535S), anti-mTOR (2972S), and anti-p-mTOR
(5536S) were obtained from Cell Signaling Technology (Beverly, MA,
USA). Anti-DDiT4 (ab106356) was purchased from Abcam (Cambridge, MA,
USA).
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