Ab26056

Manufactured by Abcam

Sourced in United States

Ab26056 is a primary antibody that can be used for the detection of a specific target protein in various laboratory applications. This antibody is produced by Abcam and is designed for research purposes.

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Lab products found in correlation

8 protocols using ab26056

Immunoblotting Analysis of Hedgehog Signaling in HCC

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Immunoblotting was exercised to examine the expression of Hh signaling pathway-related markers in HCC cells according to standard protocols as described previously.18 (link) Protein was harvested using radio immunoprecipitation assay lysis buffer, and a bicinchoninic acid assay was applied to determine the protein concentration. Next, we separated equal amount of protein using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred the protein to a polyvinylidene difluoride membrane. After blocking with 1.5% skimmed milk for 1 h at room temperature, membranes were incubated with the primary antibodies at 4°C overnight and with horseradish peroxidase-conjugated secondary antibodies. Membranes were developed using a chemiluminescent substrate. The antibodies were used as follows: Gli1 (1:200, ab49314, Abcam), Gli2 antibodies (1:500, ab26056) and Gli3 antibodies (1:500, ab6050) and the secondary antibodies (1:50,000, ab7090).

Wang Z., Song L., Ye Y, & Li W. (2020). Long Noncoding RNA DIO3OS Hinders Cell Malignant Behaviors of Hepatocellular Carcinoma Cells Through the microRNA-328/Hhip Axis. Cancer Management and Research, 12, 3903-3914.

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Immunohistochemical Analysis of Hedgehog Pathway

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Immunohistochemistry (IHC) was performed on 4-μm TMA sections using a Ventana BenchMark XT Staining systems (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. The primary antibodies used are as follows: anti-GLI1 (Cell signaling, #3538), anti-GLI2 (Abcam, ab26056), anti-PTCH1 (Abcam, ab53715), anti-PTCH2 (Abcam, ab238338), anti-SHH (Abcam, ab53281), and anti-SMO (Abcam, ab236465). GLI1 and GLI2 were evaluated for cytoplasmic and nuclear stain, while PTCH1, PTCH2, SHH, and SMO were evaluated for cytoplasmic stain. IHC was scored from 0 to 3 according to the stain intensity because a majority of cases showed a diffuse staining pattern.

Kim H.S., Kim Y.S., Lee C., Shin M.S., Kim J.W, & Jang B.G. (2019). Expression profile of sonic hedgehog signaling-related molecules in basal cell carcinoma. PLoS ONE, 14(11), e0225511.

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Western Blot Analysis of Hedgehog Pathway

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Total protein extracts were harvested and subjected to WB as described previously [23 (link)]. Primary antibodies against the following proteins were used for WB: GLI2 (ab26056; Abcam), PTCH1 (ab55629; Abcam), FOXM1 (sc-376,471; Santa Cruz Biotechnology, CA, USA), TPX2 (12,245; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore, Billerica, MA, USA). This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, namely normal goat anti-mouse IgG (31,430; Thermo Scientific Pierce) or normal goat anti-rabbit IgG (31,460; Thermo Scientific Pierce), and the membranes were probed with SuperSignal™ West Femto Maximum Sensitivity Substrate ECL (34,095; Thermo Fisher Scientific Inc). The immunoblot films were digitalized with Epson V700 scanner, and intensity of major bands were quantitated using Image J (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least thrice.

Wang Y., Wang H., Yan Z., Li G., Hu G., Zhang H., Huang D., Wang Y., Zhang X., Yan Y., Lu Q., Cheng M, & Luo S. (2020). The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation. Cell Communication and Signaling : CCS, 18, 116.

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Western Blot Analysis of Ciliary Proteins

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Collected small vesicles were resolved in 1.5x SDS sample buffer. Cultured cells were also lysed with the same buffer. Extracted proteins were denatured at 95 °C for 5 min. Proteins were separated with SDS-PAGE, and transferred to PVDF membrane (Millipore). The membrane was blocked with 10% goat serum or 5% bovine serum albumin. The blocked membrane was incubated with primary antibodies in the blocking solution at 4 °C overnight. Primary antibodies used were as follows: rabbit anti-Ift88 (1:2000; ProteinTech, 13967-1-AP); mouse anti-Kif3A (1:2000; BD Transduction, 611508); goat anti-Gli3 (1:200; R&D Systems, AF3690); rabbit anti-Gli2 (1:1000; Abcam, ab26056); rabbit anti-Sufu (1:2500; as reported in (Humke et al., 2010 (link))); rabbit anti-Arl13b (1:1000; ProteinTech, 17711-1-AP); rabbit anti-actin (1:2000; Sigma, A2066); rabbit anti-Ift122 (1:2000; ProteinTech, 19304-1-AP); rabbit anti-Ift140 (1:2000; ProteinTech, 17460-1-AP); mouse anti-α-tubulin (1:10000; Sigma, T9026); mouse anti-GAPDH (1:2000; Millipore, MAB374). The primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno Lab). Signals were developed with enhanced chemiluminescent substrate (GE) and detected with a cooled CCD camera system, LAS-3000 mini (Fuji film). Band intensities were quantified with free software, ImageJ.

Phua S.C., Chiba S., Suzuki M., Su E., Roberson E.C., Pusapati G.V., Setou M., Rohatgi R., Reiter J.F., Ikegami K, & Inoue T. (2017). Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell, 168(1-2), 264-279.e15.

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Evaluating GANT61-Cisplatin Cytotoxicity in Cells

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GANT61 purchased from Sigma-Aldrich (St. Louis, MO, USA) was dissolved in DMSO and stored at −20 °C. Cisplatin was obtained from Sigma-Aldrich and was dissolved at a stock concentration of 2 mmol/L. Rhodamine 123 was purchased from Sigma-Aldrich. Foetal bovine serum (FBS) and 0.25 % trypsin/EDTA were bought from Gibco Life Technologies (Carlsbad, CA, USA). A FITC-annexin V kit was purchased from Abcam (Cambridge, MA, USA). The CCK-8 cell count kit for cell proliferation analysis was purchased from Tongren Chemical Research Institute (Kyushu, Japan). The reverse transcription kit was purchased from Takara (Shiga, Japan). SYBR Green I was purchased from Noble Ryder (Beijing, China). Antibodies for GLI1 (ab49314), GLI2 (ab26056), Bax (ab10813), BCL-2 (ab59348), caspase-3 (ab32351) and caspase-9 (ab25758) were purchased from Abcam (Cambridge, MA, USA). The β-actin antibody (AP0060) was purchased from Bioworld (Louis Park, MN, USA).

Lin Z., Li S., Sheng H., Cai M., Ma L.Y., Hu L., Xu S., Yu L.S, & Zhang N. (2016). Suppression of GLI sensitizes medulloblastoma cells to mitochondria-mediated apoptosis. Journal of Cancer Research and Clinical Oncology, 142(12), 2469-2478.

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Western Blot Protocol for Protein Analysis

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The Western blot procedure has been previously described in detail [7 (link)]. The primary antibodies used were mouse monoclonal anti-PRR11 (1:1000) (TA800449, Origene, MD, USA), rabbit polyclonal anti-SKA2 (1:500) (PA5-20818, Invitrogen, Waltham, CA, USA), rabbit polyclonal anti-GLI1 (1:1000) (2534, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-GLI2 (1:400) (ab26056, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-NF-Y (1:1000) (sc-13045X, Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit polyclonal anti-GAPDH (1:5000) (AB-P-R001, Goodhere Biotechnology, Hangzhou, China).

Sun Y., Xu D., Zhang C., Wang Y., Zhang L., Qiao D., Bu Y, & Zhang Y. (2021). HEDGEHOG/GLI Modulates the PRR11-SKA2 Bidirectional Transcription Unit in Lung Squamous Cell Carcinomas. Genes, 12(1), 120.

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Sufu Expression Analysis in Limb Buds

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To detect the expression of Sufu in the wild type and Sufu mutant limb buds, the samples with horizontal paraffin sections were subjected to immunohistochemical analysis with Sufu antibody (1:200) according to the method depicted previously [35 (link)]. For immunoblot analysis, whole cell protein lysates were prepared from dissected embryonic limb buds at embryonic day 10.25 (E10.25), E10.5, and E11.5. 40 μg of protein lysates were run on a 7.5% PAGE gel and transferred onto membrane for blot analysis as described previously [35 (link)]. Sufu antibody (1:1000) and Gli2 antibody (1:1000) were purchased from Abcam (ab52913 and ab26056, respectively). Gli3 antibody (1:1000) was from R&D System (AF3690). The gray values of immunoblot bands were evaluated with Image-Pro Plus software (version 6.0). The relative expression level of a protein was calculated by dividing its gray value by that of the corresponding reference protein. The significance of expression differentiation between wild type and mutant samples was analyzed with Student’s t test.

Li J., Wang Q., Cui Y., Yang X., Li Y., Zhang X., Qiu M., Zhang Z, & Zhang Z. (2015). Suppressor of Fused Is Required for Determining Digit Number and Identity via Gli3/Fgfs/Gremlin. PLoS ONE, 10(5), e0128006.

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Hedgehog Signaling Pathway Analysis

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Liver tissues were homogenized and digested in 1×RIPA buffer containing a protease inhibitor and phosphatase inhibitor cocktail solution (Thermo Scientific, Waltham, MA, USA). Western blotting experiments were performed following a standard protocol. The primary antibodies used were anti-SHH (SC-9024; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Gli1 (ab167388; Abcam, Cambridge, UK), anti-Gli2 (ab26056; Abcam, Cambridge, UK), and anti-GAPDH (# 2118; Cell Signaling Technology, Danvers, MA, USA).

Cho K., Moon H., Seo S.H., Ro S.W, & Kim B.K. (2021). Pharmacological Inhibition of Sonic Hedgehog Signaling Suppresses Tumor Development in a Murine Model of Intrahepatic Cholangiocarcinoma. International Journal of Molecular Sciences, 22(24), 13214.

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