Rabbit anti lkb1

Manufactured by Cell Signaling Technology

Sourced in Switzerland

The Rabbit anti-LKB1 is a primary antibody that recognizes the LKB1 (Liver Kinase B1) protein. LKB1 is a serine/threonine protein kinase that plays a critical role in cellular energy regulation and metabolism.

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Lab products found in correlation

Mouse anti actin, by Merck Group (3 mentions) Rabbit anti lc3, by Merck Group (3 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions) Immobilon western, by Merck Group (2 mentions) Rabbit anti ift20, by Proteintech (2 mentions) Secondary hrp conjugate anti rabbit igg, by GE Healthcare (2 mentions) Anti mouse igg hrp conjugate, by Bio-Rad (2 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) E7 antibody, by Developmental Studies Hybridoma Bank (1 mentions) Rabbit anti fak, by Cell Signaling Technology (1 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) Mouse anti beclin1, by BD (1 mentions) Rabbit anti rab11, by Cell Signaling Technology (1 mentions) Rabbit anti fip200, by Merck Group (1 mentions) Rabbit anti ift88, by Proteintech (1 mentions) Mouse anti α tubulin, by Merck Group (1 mentions) Rab11a, by Thermo Fisher Scientific (1 mentions) Claudin 3, by Thermo Fisher Scientific (1 mentions) Antibodies against claudin 1, by Thermo Fisher Scientific (1 mentions) Anti phospho ampk thr172, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Anti-LKB1 (18 citations)

8 protocols using rabbit anti lkb1

Western Blot Analysis of Key Signaling Proteins

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Cells were lysed in 1X lysis buffer (Cell Signaling) and 30 ug of cell lysates were resolved by 10% SDS-PAGE gel and transferred overnight to a PDVF membrane at 0.07A at 4°C. Membranes were blocked in 5% BSA or 5% milk for one hour at room temperature and then incubated overnight at 4°C with rabbit anti-HD-PTP (1:1,000, Bethyl Laboratories), mouse anti-Integrinβ1/CD29(1:1,000 BD Biosciences), rabbit anti-phospho FAK(Tyr397) (1:1,000, Invitrogen), rabbit-anti-FAK(1:1,000, Cell Signaling) and rabbit anti-LKB1 (1:1000, Cell Signaling) in TBS-T (0.1% Tween 20) containing 5% BSA or 3% milk. Tubulin was detected as a loading control by incubation of the membrane with the E7 antibody from Developmental Studies Hybridoma Bank at a 0.2 ug/ml dilution for overnight at 4°C. Membranes were washed in TBS-T, incubated overnight with primary antibodies at 4 degrees and detected with goat anti-rabbit or mouse-HRP secondary antibodies (Thermo Scientific) followed by detection using SuperSignal^™ West Pico Plus chemiluminescent substrate (Thermo Scientific).

Seong C.S., Huang C., Boese A.C., Hou Y., Koo J., Mouw J.K., Rupji M., Joseph G., Johnston H.R., Claussen H., Switchenko J.M., Behera M., Churchman M., Kolesar J.M., Arnold S.M., Kerrigan K., Akerley W., Colman H., Johns M.A., Arciero C., Zhou W., Marcus A.I., Ramalingam S.S., Fu H, & Gilbert-Ross M. (2023). Loss of the endocytic tumor suppressor HD-PTP phenocopies LKB1 and promotes RAS-driven oncogenesis. bioRxiv.

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Western Blot Analysis of Autophagy Proteins

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Cells in microslides were washed twice with ice-cold PBS and lysed in ice with 150 µl of 1X Laemmli buffer (60 mM Tris-HCL pH = 6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95 °C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (1:1000, Sigma, Cat#L7543); mouse-anti-actin (1:5000, Millipore, Cat#1501); mouse-anti-Beclin1 (1:2000, BD Biosciences,Cat#612113); rabbit-anti-IFT88 (1:1000, Proteintech, Cat#13967-1-AP); rabbit-anti-PIK3C2α (1:800, Novus, Cat#NBP2-19829); mouse-anti-Wipi2 (2A2) (1:1000, AbD Serotec, Cat#MCA5780GA); rabbit-anti-Rab11 (1:1000, Cell Signaling, Cat#2413S); rabbit-anti-VPS34 (d9A5) (1:500, Cell Signaling, Cat#4263); rabbit-anti-LKB1 (1:1000, Cell signaling, Cat#3050); rabbit-anti-FIP200 (1:1000, Sigma, Cat#SAB4200135); mouse-anti-ATG16L1 (1:1000, MBL, Cat#PM040); rabbit-anti-IFT20 (1:1500, Proteintech, Cat#13615). Secondary HRP conjugate anti‐rabbit IgG (GE Healthcare) and HRP conjugate anti‐mouse IgG (Bio‐Rad).

Boukhalfa A., Nascimbeni A.C., Ramel D., Dupont N., Hirsch E., Gayral S., Laffargue M., Codogno P, & Morel E. (2020). PI3KC2α-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress. Nature Communications, 11, 294.

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Immunostaining of Tight Junction Proteins

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The rat monoclonal antibody against ZO‐1 clone R40.76 was used.21 Antibodies against claudin‐1 (catalog no. 71‐7800), claudin‐3 (catalog no. 34‐1700), Rab11a (catalog no. 71‐5300) and rhodamine phalloidin were obtained from Thermo Fisher Scientific (Waltham, MA). The rabbit polyclonal antibody against Par‐3 was obtained from Milipore (catalog no. 07‐330; Billerica, MA). The rabbit polyclonal antibody against Bsep was obtained from Kamiya Biomedical Company (catalog no. MC‐333; Tukwila, WA). The rabbit antibody against cingulin was a gift from Sandra Citi (University of Geneva, Geneva, Switzerland),22 and the Myosin Vb was a gift from John Hammer (NHLBI, NIH).23 Rabbit anti‐LKB1 (catalog no. CST3047), anti‐AMPK (catalog no. CST2532), and anti‐phospho‐Thr172 AMPK (catalog nos. pAMPK and CST2535) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Mouse anti‐α‐tubulin (catalog no. T6199) was obtained from Sigma‐Aldrich.

Porat‐Shliom N., Tietgens A.J., Van Itallie C.M., Vitale‐Cross L., Jarnik M., Harding O.J., Anderson J.M., Gutkind J.S., Weigert R, & Arias I.M. (2016). Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo. Hepatology (Baltimore, Md.), 64(4), 1317-1329.

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AMPK Purification and Phosphorylation

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Recombinant His-tagged AMPK complexes were expressed in Escherichia coli and purified by chromatography on nickel-Sepharose [9 (link)]. AMPK was phosphorylated on Thr¹⁷² by overnight incubation with MgATP and CaMKKβ, and repurified as previously described [9 (link)]. Subsequent incubation with MgATP and CaMKKβ did not increase Thr¹⁷² phosphorylation, demonstrating that Thr¹⁷² is maximally phosphorylated using these conditions. The following antibodies were from Cell Signaling: rabbit anti-AMPKα1 (#2795), rabbit anti-AMPKα2 (#2757), mouse anti-AMPKα1/2 (#2793), rabbit anti-AMPKβ1/2 (#4150), rabbit anti-ACC (#3676), rabbit anti-pACC (#3661), rabbit anti-AMPKγ1 (#4187), rabbit anti-LKB1 (#3050) and rabbit anti-pThr¹⁷² (#2535). Mouse anti-vinculin was from Sigma–Aldrich (V9131). Mouse monoclonal anti-CaMKKβ antibody was a generous gift from Prof. Grahame Hardie (Dundee University).

Willows R., Sanders M.J., Xiao B., Patel B.R., Martin S.R., Read J., Wilson J.R., Hubbard J., Gamblin S.J, & Carling D. (2017). Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells. Biochemical Journal, 474(17), 3059-3073.

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Western Blot Analysis of Cell Signaling Proteins

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Cells in microslides were washed twice with ice-cold PBS and lysed on ice with 150 μl of 1X Laemmli buffer (60 mM Tris-HCl pH=6.8, 2% SDS, 10% Glycerol, bromophenol blue, supplemented with 100 mM DTT) for 30 min. Samples were boiled for 10 min at 95°C, separated by SDS/PAGE and then transferred onto Nitrocellulose membranes. Western blot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore). The following antibodies were used in immunoblotting: rabbit-anti LC3 (Sigma, Cat#L7543); rabbit-anti-FLCN (Cell signaling, Cat#3697); rabbit-anti-FNIP1 (Abcam, Cat#ab134969); rabbit-anti-AMPK (Cell signaling, Cat#2532S); rabbit-anti-p-AMPK (T172) (Cell signaling, Cat#2535); mouse-anti-actin (Millipore, Cat#1501); rabbit-anti-ATG16L1 (MBL, Cat#PM040); rabbit-anti-IFT20 (Proteintech, Cat#13615-1-AP); rabbit-anti β-catenin (Cell signaling, Cat#8480); rabbit-anti-LKB1 (Cell signaling, Cat#3050); rabbit-anti-S6 ribosomal protein (Cell signaling, Cat#2217); rabbit-anti-p-S6 ribosomal protein (S240/244) (Cell signaling, Cat#2215); rabbit-anti-Tuberin/TSC2 (Cell signaling, Cat#4308); rabbit-anti-p-Tuberin/TSC2 (T1462) (Cell signaling, Cat#3617). Secondary HRP conjugate anti-rabbit IgG (GE Healthcare) and HRP conjugate anti-mouse IgG (Bio-Rad).

Zemirli N., Boukhalfa A., Dupont N., Botti J., Codogno P, & Morel E. (2019). The primary cilium protein folliculin is part of the autophagy signaling pathway to regulate epithelial cell size in response to fluid flow. Cell Stress, 3(3), 100-109.

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Quantification of Autophagy Markers

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Cytosolic and nuclear proteins were extracted from heart tissue samples by using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer. After protein concentration was measured, equal amounts of cytosolic and nuclear proteins were electrophoresed, blotted, and incubated with antibodies and developed. Equal amount of protein lysates from each heart tissue was added to each well, and β-actin was utilized as a protein marker for normalization. The antibodies used above were listed: rabbit anti-LC3 (Sigma Cat#:L8918), rabbit anti-ATG5 (Cell Signaling Technology Cat#:2630), rabbit anti-ATG14 (Cell Signaling Technology Cat#:5504), rabbit antiphospho-ATG14 (Ser29) (Cell Signaling Technology Cat#:92340), rabbit anti-AMPKα (Cell Signaling Technology Cat#:5832), rabbit antiphospho-AMPKα (Thr172) (Cell Signaling Technology Cat#:2535), rabbit anti-ULK1 (Cell Signaling Technology Cat#:6439), rabbit antiphospho-ULK1 (Ser555) (Cell Signaling Technology Cat#:5869), rabbit anti-LKB1 (Cell Signaling Technology Cat#:3050S), rabbit anti-p62/SQSTM1 (MBL Cat#:PM045), rabbit anti-ATG7 (Cell Signaling Technology Cat#:8558), mouse anti-GAPDH (Sigma Cat#:ab181602), goat antimouse IgG (H+L), Alexa Fluor 546 (Thermo Fisher Scientific Cat #: A-11003), goat antirabbit IgG (H+L), Alexa Fluor 546 (Thermo Fisher Scientific Cat #: A-11010).

Chen J., Miao X., Liu C., Liu B., Wu X., Kong D., Sun Q, & Gong W. (2020). BET Protein Inhibition Prolongs Cardiac Transplant Survival via Enhanced Myocardial Autophagy. Transplantation, 104(11).

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Quantifying Mitochondrial Proteins in LKB1 Knockout Mice

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Hippocampal regions were dissected from 3-wk-old wild-type (NEX^Cre;LKB1^F/+ or NEX^Cre;LKB1^+/+) or Nex^Cre;LKB1^F/F conditional knockout (KO) mice and lysed with RIPA buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl in 50 mM Tris buffer (pH 8.0) supplemented with the cocktail of protease inhibitors (Roche). Samples were loaded onto SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane (Amersham). After transfer, membranes were blocked for 30 min with blocking buffer containing 5% skim milk in TBS-T (20 mM Tris-HCl [pH7.4], 150 mM NaCl, 0.1% Tween 20). Then, primary antibodies were incubated overnight at 4°C, and washed three times by TBS-T. HRP-coupled secondary antibodies (Invitrogen) were incubated for 1 h at room temperature followed by three times washes with TBS-T. Chemiluminescence images were taken by Fluorochem Q imager (ProteinSimple) and quantified using AlphaView software (ProteinSimple). Primary antibodies are rabbit anti-MCU (1:2000, Sigma), mouse anti-OxPhos complex kit (1:2000, Invitrogen), rabbit anti-LKB1 (1:1000, Cell Signaling), and mouse anti-actin (1:5000, Millipore).

Kwon S.K., Sando R I.I.I., Lewis T.L., Hirabayashi Y., Maximov A, & Polleux F. (2016). LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons. PLoS Biology, 14(7), e1002516.

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Immunostaining of Cilia-Associated Proteins

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Cells were seeded and grown on coverslips to confluence. Then, cells were incubated for 24 h in opti-MEM to induce ciliary expression, and finally washed and fixed with ice-cold methanol, and permeabilized with PBS Tween 0.1%. After blocking cells were incubated with primary antibodies rabbit anti LKB1 (1:100; Cell Signaling, Danvers, MA), anti-mouse acetylated alpha tubulin (1:200; Sigma), anti-mouse gamma tubulin (1:100; Sigma), and ARL13b (1:100; Santa Cruz, Dallas, TX) overnight at 4°C, washed and incubated with Alexa Fluor 594 conjugated goat anti-rabbit and Alexa Fluor 488 conjugated goat anti-mouse (1:100 Life Technologies, Carlsbad, CA) for 1 h. The nuclei were visualized with DAPI and the immunostainings were viewed and documented using a Zeiss Axio Observer inverted microscope. At least 200 cells were analyzed from each sample and the images were taken at a 630X magnification.

Mansini A.P., Peixoto E., Jin S., Richard S, & Gradilone S.A. (2019). The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth. Hepatology (Baltimore, Md.), 69(4), 1582-1598.

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