Cells were stained using Abs specific for mouse Ags: CD45R (B220) (various fluorochromes) (BD Pharmingen, BioLegend, San Diego, CA and Tonbo Biosciences); IgM (various fluorochromes, Jackson ImmuoResearch Laboratories, West Grove, PA); CD19 eFlour450, CD25 APC, MHC II APC (Tonbo Biosciences); CD43 PE, BP-1 PE, CD117 PE, CD24 (HSA) PE-Cy7, IgD FITC (BD Pharmingen); CD21 PerCP/Cy5.5 (BioLegend). IC staining was performed with IC fixation and permeabilization buffer (eBiosciences). Methanol fixation was performed for IC phospho (p)-S6R detection. Abs used for IC staining were p-ribosomal S6 protein (S6R) S235/236 PE (eBiosciences); p-AMPKα T172, c-Myc AF488 and p-Ulk1 S555 (Cell Signaling Technology, Danvers, MA). Donkey anti-rabbit Alexa-Fluor 647 (Life Technologies, Carlsbad CA) secondary Ab was used to detect unlabeled primary Abs. Data was collected using FACS Canto II or LSR II flow cytometers (BD Biosciences) and analyses were performed using FlowJo software (TreeStar, Ashland, OR).
P ulk1s555
Manufactured by Cell Signaling Technology
Sourced in United States
P-ULK1S555 is an antibody product offered by Cell Signaling Technology. It is designed to detect endogenous levels of ULK1 protein phosphorylated at serine 555.
Automatically generated - may contain errors
Lab products found in correlation
P ampkαt172, by Cell Signaling Technology (3 mentions)
Paccs79, by Cell Signaling Technology (3 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Ps6 s235 236, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Facscanto 2, by BD (1 mentions)
Cd45r b220, by BD (1 mentions)
Ic fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Igd fitc, by BD (1 mentions)
Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Cd21 percp cy5, by BioLegend (1 mentions)
Cd117 pe, by BD (1 mentions)
Ampk alpha, by Cell Signaling Technology (1 mentions)
Paxillin, by BD (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Total acc, by Cell Signaling Technology (1 mentions)
P p90rsks380, by Cell Signaling Technology (1 mentions)
Phospho fak y397, by Abcam (1 mentions)
Wnt5a b, by Cell Signaling Technology (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
8 protocols using p ulk1s555
1
Comprehensive Flow Cytometry Immunophenotyping
2
Signaling Pathway Antibody Analysis
3
Cucurbitacin E Mediated Autophagy Regulation
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Cucurbitacin E was obtained from Shanghai Shunbo Chempharm Co (Shanghai, China). Chloroquine (CQ), dimethylsulfoxide (DMSO) and sodium dodecyl sulfate (SDS), polyformaldehyde were from Sigma-Aldrich (St. Louis, MO, USA). WST-1 was from Roche (Penzberg, Germany). Lipofectamine RNAiMAX, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, L-glutamine, and fetal bovine serum (FBS) were from Gibco/ Invitrogen (Carlsbad, CA, USA). Polyvinylidene difluoride (PVDF) membranes (Hybond-P) were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Antibodies against p-p70S6KT389, p70S6K, p-4E-BP1T37/46, 4E-BP1, p-S6S235/236, S6, p62/SQSTM1, LC3B, ATG5, Beclin 1, p-AKTS473, p-AktT308, Akt, p-AMPKαT172, AMPKα, p-AMPKβ1S108, AMPKβ1, p-ULK1S758, p-ULK1S555, ULK1, p-RaptorS792, Raptor, β-tubulin and HRP-conjugated second antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against LAMP2 was obtained from Abcam (Cambridge, MA, USA).
4
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from tissue or cells using cell lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 100 mM NaF, 10 Napyrophosphate, 5 EDTA mM, 250 mM sucrose, 1 mM DTT, and 1 mM Na-orthovanadate, 1% Triton X and Complete protease inhibitor cocktail (Roche)). Samples (1 μg/μL) were prepared in 4× SDS sample buffer and boiled at 95 °C for 5 min. Sample proteins were separated in a 10% or 12% SDS–PAGE gel and transferred to a PVDF membrane at 100 V for 90 min. Membranes were blocked with 5% skim milk in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.05 % Tween 20) for 1 h. Subsequently, membranes were subject to primary antibody (1:1000 dilution) in TBST (with 5% BSA) overnight at 4 °C and then incubated with secondary antibody (1:1000 dilution) at room temperature for 1 h. Protein bands were imaged using electrochemiluminescence and analyzed using Image J software (National Institutes of Health, Bethesda, USA). Antibodies against pACCS79 (#3661), ACC (#3662), pAMPKαT172 (#2535), AMPKα (#2532), pULK1S555 (#5869), ULK1 (#8054), p-HSLS660 (#4126), HSL (#4107), LC3B (#2775), p38 (#8690), p-p38 (#9211), p62 (#5114), pPKA substrate (#9621) and p62 (#5114) were purchased from Cell Signaling (Danvers, MA). Antibody against UCP1 (#UCP11-A) was purchased from Alpha Diagnostic International. Antibody against β-tubulin (#32-2600) was purchased from Invitrogen.
5
Immunohistochemical Analysis of Autophagy Markers
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Paraffin-embedded tissue sections were prepared as described previously (Guo et al., 2013 (link)) for H and E, and IHC staining. Antibodies utilized for IHC were Atg7 (Sigma Aldrich, A2856, RRID:AB_1078239 ), Lkb1 (Santa Cruz Biotechnology, sc-32245, RRID:AB_627890 ), p62 (Enzo Life Sciences, PW9860-0100, RRID:AB_2877676 ), p-AMPKTh172 (Cell Signaling, 2535S, RRID:AB_331250 ), p-ACCS79 (Cell Signaling, 3661, RRID:AB_330337 ), p-S6S235/236 (Cell Signaling, 4858, RRID:AB_916156 ), p-ULK1S555 (Cell Signaling, 5869, RRID:AB_10707365 ), p-ULK1S757 (Cell Signaling, 14202, RRID:AB_2665508 ), LC3 (Nano Tools, LC3-5F10, RRID:AB_2722733 ), Ki67 (Abcam, ab-15580, RRID:AB_443209 ), cleaved caspase-3 (Cell Signaling, 9661S), OLFM4 (Cell Signaling, 39141, RRID:AB_2650511 ), lysozyme (Agilent, A0099, RRID:AB_2341230 ), and p53 (Novus Biologicals, NB200-103SS, RRID:AB_2877680 ). Paraffin embedded tissue sections were used for the TUNEL assay by means of the HRP-DAB TUNEL staining kit (ab206386) and the slides were counterstained by methyl blue following the protocol provided by the TUNEL staining kit. For the quantification of IHC and TUNEL assay, tissues were analyzed by quantifying at least 10 images at 20x magnification. A minimum of 200 cells were scored for each image.
6
Western Blot Analysis of Cardiac Tissue
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The ischemic zone of the left ventricle (LV) heart tissue was snap frozen and ground with mortar and pestle in liquid nitrogen. The powdered tissue was then suspended in RIPA lysis buffer.
29 (link) H9c2 cells were used for Western blot analysis and cells were suspended in lysis buffer. Proteins were separated by reducing SDS‐PAGE and transferred to a PVDF membrane. The following antibodies were used for Western blot: p‐AMPK T172 (1:1000, Cell Signaling, #2535), p‐ULK1 S555 (1:1000, Cell Signaling, #5869), GAPDH (1:2000, ThermoFisher, #MA5‐15738), Adiponectin (1:1000, ImmunoDiagnostics, #12010), ATG5‐12 (1:1000, Novus Biologicals, #NB110‐53818), ATG7 (1:1000, Santa Cruz, #sc‐376212), Beclin1 (1:1000, Cell Signaling, #3738), LC3B (1:1000, Cell Signaling, #2775), p62 (1:1000, Cell Signaling, #5114), BAX (1:1000, Cell Signaling, #2772), cleaved Caspase 3 (1:1000, Cell Signaling, #9661), bcl‐2 (1:1000, Cell Signaling, #3489), and β‐actin (1:2000, ThermoFisher, #PA1‐46296) were used. The quantification of signals was performed by densitometry of scanned autoradiographs with the aid of ImageJ (version 1.4v).
29 (link) H9c2 cells were used for Western blot analysis and cells were suspended in lysis buffer. Proteins were separated by reducing SDS‐PAGE and transferred to a PVDF membrane. The following antibodies were used for Western blot: p‐AMPK T172 (1:1000, Cell Signaling, #2535), p‐ULK1 S555 (1:1000, Cell Signaling, #5869), GAPDH (1:2000, ThermoFisher, #MA5‐15738), Adiponectin (1:1000, ImmunoDiagnostics, #12010), ATG5‐12 (1:1000, Novus Biologicals, #NB110‐53818), ATG7 (1:1000, Santa Cruz, #sc‐376212), Beclin1 (1:1000, Cell Signaling, #3738), LC3B (1:1000, Cell Signaling, #2775), p62 (1:1000, Cell Signaling, #5114), BAX (1:1000, Cell Signaling, #2772), cleaved Caspase 3 (1:1000, Cell Signaling, #9661), bcl‐2 (1:1000, Cell Signaling, #3489), and β‐actin (1:2000, ThermoFisher, #PA1‐46296) were used. The quantification of signals was performed by densitometry of scanned autoradiographs with the aid of ImageJ (version 1.4v).
7
Analysis of Cellular Signaling Pathways
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Antibodies against LKB1, pLKB1S428, AMPK, pAMPKThr172, p70S6K, pp70S6KThr389, S6 Ribo, pS6 RiboS235/236, pS6 RiboS240/244, ULK1, pULK1S555, pULKS757, LC-3B, cyclin A, cyclin D1, and PARP were purchased from Cell Signaling. Antibodies for p27, cyclin B1, and CDC25C were from Santa Cruz Biotechnology, and β-actin from Sigma-Aldrich. Secondary antibodies were purchased from GE Healthcare. Cells were treated with indicated concentrations of hMGL-4.0 for the specified time. After incubation, cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer. Proteins were separated by using 4 to 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking in 5% BSA for 1 h, the membranes were probed with specific primary antibodies (listed above) overnight at 4 °C. Following secondary antibody incubation, the membranes were visualized using a commercially available chemiluminescent detection kit (Pierce Biotechnology).
8
Antibody-based Protein Expression Analysis
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Antibodies against p62 (#610832) were purchased from BD Pharmingen. Antibodies against GAPDH (#5174), LKB1 (#3050S), mTOR (#2972), p-mTOR (S2448; #2971S), AMPKα (#2603), p-AMPK (T172; #2535), ACC (#3676), p-ACC (S79; #3661), ULK1 (#8054) and p-ULK1 (S555; #5869) were purchased from Cell Signaling. Polyclonal antibodies against OGT (#SAB2101676) and OGA (#SAB4200311) were purchased from Sigma-Aldrich. The monoclonal antibody against O-GlcNAc (RL2; #MA1072) was purchased from Thermo Fisher Scientific. Antibodies against LC3 (#NB100-2220SS) were purchased from Novus Biological. The anti-GFP monoclonal antibody (#SC9996) was purchased from Santa Cruz Biotechnology. DON, TG and bafilomycin A1 (Baf A1) were purchased from Sigma-Aldrich. The pcDNA6.2-myc construct containing AMPKα, OGT, OGA or null control and the pLKO shRNA construct containing OGT, OGA, AMPKα or negative control were purchased from Addgene.
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