Ab111344

Manufactured by Abcam

Ab111344 is a recombinant monoclonal antibody that recognizes the human ABCB1 protein. ABCB1 is a member of the superfamily of ATP-binding cassette (ABC) transporters. It functions as an ATP-dependent drug efflux pump for a variety of substrates.

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Lab products found in correlation

7 protocols using ab111344

Phospho-STAT3 T622 Antibody Production

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Rabbit polyclonal antibody recognizing phosphorylated STAT3 T622 was customized from Boer Biotechnology. To prepare antibody recognizing STAT3 pT622, rabbits were treated with peptide containing STAT3 pT622. Nonmodified peptide immobilized on an affinity column was used to remove the antibodies recognizing nonphosphorylated STAT3, and STAT3 pT622 peptide immobilized on an affinity column was used to associate with and isolate the antibodies. The eluted antibodies were then concentrated.
Antibodies recognizing TAZ (#83669), STAT3 (#9139), MST2 (#3952), MST1 (#14946), YAP1 (#12395), SAV1 (#13301), and STAT3‐pY705 (#9145) were obtained from Cell Signaling Technology. Antibody simultaneously recognizing LATS1 and LATS2 (BS‐4081R) was obtained from Bioss. Antibodies recognizing LATS1/2 pT1079/T1041 (ab111344), Ki67 (ab16667), YAP1 (ab205270), HA (ab9110), tubulin (ab7291), Flag (ab205606), GST (ab36415), YAP1 pS127 (ab76252), phospho‐Thr (ab9337), JAK2 (ab108596), and recombinant IL‐6 protein (ab9627) were purchased from Abcam. Anti‐Flag agarose beads were obtained from Sigma. XMU‐MP‐1 was obtained from MedChemExpress. [γ‐³²P]‐ATP was obtained from MP Biomedicals.

Tang Q., Fang J., Lai W., Hu Y., Liu C., Hu X., Song C., Cheng T., Liu R, & Huang X. (2022). Hippo pathway monomerizes STAT3 to regulate prostate cancer growth. Cancer Science, 113(8), 2753-2762.

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Immunohistochemical Analysis of Tumor Markers

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Formalin-fixed, paraffin-embedded (FFPE) tissue samples were sliced in consecutive 3.0-μm-thick sections, which were dewaxed in xylene and rehydrated in graded ethanol. Immunohistochemical staining was then performed as per the Dako REAL EnVision Detection System (K5007, Dako) manual. The following primary antibodies were used:
Anti-LATS1/2 (1:100; ab111344, Abcam), Anti-CD163 (1:100, ab87099, Abcam), Anti-CD8 (1:100, ab4055, Abcam), Anti-FOXP3 (1:100, ab20034, Abcam), Anti-MLH1 (1:50, clone ES05, DAKO), Anti-PMS2 (1:40, clone EP51, DAKO), Anti-MSH2 (1:50, clone FE11, DAKO), and Anti-MSH6 (1:50, clone EP49, DAKO).
Paraffin-embedded sections (3.0 μm) were prepared for immunohistochemical analyses. After deparaffinization, all antigens except nestin were retrieved at 120°C for 15 min in a sodium citrate buffer solution (pH 6.0). Tissues were incubated with 0.3% hydrogen peroxide for 30 min and then blocked with 1% bovine serum albumin (Sangon, Shanghai, China) overnight at 4°C. The peroxidase reaction was developed using a 3,3-diaminobenzidine (DAB) chromogen solution in a DAB buffer substrate and then counterstained with hematoxylin.

Guo Y., Liu X., Xu D., Huang C., Wang Z., Xia X., Zhu C., Xu J., Zhang Z., Shen Y., Zhao W, & Zhao G. (2020). Role of LATS1/2 in Prognosis of Advanced Gastric Cancer and Its Relationship With the Tumor Immune Microenvironment. Frontiers in Oncology, 10, 1406.

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Immunoblotting Analysis of Gastric Cancer Cells

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MKN28 and AGS gastric cancer cells were lysed using RIPA lysis buffer (Beyotime, Beijing, China) and protease inhibitors (Roche, CA, USA). The proteins were separated by 10% SDS polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose (NC) membranes. The membranes were blocked in TBS buffer containing 5% skim milk for 1 h at room temperature. After incubation with primary and secondary antibodies, ECL reagent was used to visualize the protein bands. CLDN6 (ab107059, Abcam), p-LATS1/2 (ab111344, Abcam), LATS1/2 (202761-AP, Proteintech), p-YAP1 (ab76252, Abcam), YAP1 (13584-1-AP 6900-1-Ig, Proteintech), snail1 (13099-1-AP, Proteintech), twist1(25465-1-AP, Proteintech), zeb1 (21544-1-AP, Proteintech), E-cadherin (20874-1-AP, Proteintech), N-cadherin (GB111009, Servicebio), Vimentin (GB11192, Servicebio), Ki67 (GB13030, Servicebio), β-actin (GB11001, Servicebio), HRP (GB23301, GB23303, Servicebio), Lamin B1 (AB0054, Abways).

Yu S., Zhang Y., Li Q., Zhang Z., Zhao G, & Xu J. (2019). CLDN6 promotes tumor progression through the YAP1-snail1 axis in gastric cancer. Cell Death & Disease, 10(12), 949.

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Western Blot Analysis of Hippo Pathway

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Total proteins were isolated from cells using RIPA buffer reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing protease inhibitor (Cocktail, Roche, Basel, Switzerland). The same amount (30 μg) of protein samples was separated by 10% SDS-PAGE (Mlbio, Shanghai, China) and transferred to nitrocellulose membranes (GE Healthcare, Westborough, MA). The membranes were blocked with 5% BSA at room temperature for 2 h, followed by incubation with primary antibodies: anti-p-LATS1 (1:1000, ab111344, Abcam), ANTI-LATS1 (1:1000, ab243656, Abcam), anti-p-YAP (1:1000, ab76252, Abcam), anti-YAP (1:1000, ab52771, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam) at 4 °C overnight. TBST buffer was used to elute the membrane for two times. The membranes were then incubated with the secondary antibody for 2 h at 37 °C. The ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA) was used to image the protein bands.

Ji L., Chen S., Gu L., Wang J, & Zhang X. (2021). LncRNA AGAP2-AS1 Promotes Cancer Cell Proliferation, Migration and Invasion in Colon Cancer by Forming a Negative Feedback Loop with LINC-PINT. Cancer Management and Research, 13, 2153-2161.

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Investigating Protein Signaling Pathways

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The transfected TPC-1 and CAL62 were lysed with RIPA buffer (Ding Guo Chang Sheng Biotech, Beijing, China) for 30 min on ice. Following centrifugation at 12,000×g, the concentrations of the cell lysates were measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The cell lysates (30 µg) were analyzed with SDS-PAGE, and electro-transferred onto the PVDF membrane (Thermo Fisher Scientific). The primary antibodies, including anti-LATS1 (ab70561) and anti-p-LATS1 (ab111344) (1:1,500; Abcam); anti-YAP (ab52771) and anti-p-YAP (ab62751) (1:2,000; Abcam); anti-LMO3 (ab230490), anti-LIMK1 (ab39641), and anti-p-LIMK1 (ab194798) (1:2,500; Abcam); anti-Bcl-2 (ab194583) and anti-β-actin (ab8227) (1:3,000; Abcam); anti-cleaved caspase-3 (ab2302) and anti-cleaved PARP (ab4830) (1:3,500; Abcam); anti-cofilin (ab42824) and anti-p-cofilin (ab12866) (1:4,000; Abcam); anti-β-catenin (ab6302), anti-β-tubulin (ab6046), and anti-Histone H3 (ab18521) (1:4,500; Abcam); were used to probe the membranes that were blocked with 5% bovine serum albumin. The membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (ab6721) (1:6,000; Abcam), and the blots were detected by enhanced chemiluminescence (KeyGen, Nanjin, China).

Ling Z., Long X., Wu Y., Li J, & Feng M. (2022). LMO3 promotes proliferation and metastasis of papillary thyroid carcinoma cells by regulating LIMK1-mediated cofilin and the β-catenin pathway. Open Medicine, 17(1), 453-462.

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Immunoblotting Cell Lysate Protocol

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Cell lysate for immunoblotting was performed according to the standard protocol. Briefly, cells were lysed with buffer that freshly added protease and phosphatase inhibitor cocktail (B15001, Bimake); then, 4%–20% SDS polyacrylamide gels was used to separate proteins, following transferred onto 0.22-μm NC membranes (Thermo Fisher Scientific), and incubated with primary antibodies at 4 °C overnight. The next day, HRP-conjugated secondary antibodies were incubated at room temperature (RT) for 2 h. β-actin was used for the total protein loading control. The primary antibodies and dilutions as follows: GPR4(1:1000, ab75330, Abcam), Phospho-MST1 (T183) (1:1000, 49,322, Cell signalling), MST1(1:1000, 3682, Cell signalling), Phospho-LATS1/2(T1079 + T1041)(1:1000, ab111344, Abcam), Phospho-YAP1 (S127) (1:1000, 13,088, Cell signalling), YAP1(1:1000, 14,074, Cell signalling), β-actin (1:3000, ab8226, Abcam).

Yu M., Cui R., Huang Y., Luo Y., Qin S, & Zhong M. (2019). Increased proton-sensing receptor GPR4 signalling promotes colorectal cancer progression by activating the hippo pathway. EBioMedicine, 48, 264-276.

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Western Blot Profiling of Hippo Pathway Proteins

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In brief, the cells were washed with PBS three times and lysed and denatured in RIPA lysate (Beyotime Biotechnology, China), with protease inhibitor for 30 min. The protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membrane was blocked with TBST (T1085-500, Solarbio) containing 5% skimmed milk for 2 h and then incubated with primary antibodies including: anti-MST1 (0.2 µg/mL, ab245826, Abcam), anti-p-MST1 (1/500, ab79199, Abcam), anti-LATS1/2 (1/100, orb193134, Biobyt), anti-p-LATS1/2 (1/100, ab111344, Abcam), anti-YAP (1/5,000, ab52771, Abcam), anti-p-YAP (1/10,000, ab76252, Abcam), anti-TAZ (1 μg/mL, ab84927, Abcam), anti-p-TAZ (1/200, sc17610, Santa Cruz), and anti-GAPDH (1/2,500, ab9485, Abcam) overnight at 4°C. After being washed with PBS twice, HRP-conjugated secondary antibodies (1:5,000, Abcam) were incubated with the membranes at room temperature for 1 h. The diluent for secondary antibodies was TBST buffer. The membranes were detected using an ECL western blot kit (K820500, Biovision Inc., USA) and analyzed by ImageJ.

Li Y., Xu Z, & Chang S. (2021). Glucocorticoids induce osteonecrosis of the femoral head through the Hippo signaling pathway. Open Life Sciences, 16(1), 1130-1140.

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