Proteins were extracted from bone tissue, and immunoblotting was carried out as we described previously 31 (link). Immunoblotting was performed with the primary antibodies against Shh (ab135240; Abcam, Cambridge, MA, USA), Bmi1 (10832-1-AP; Proteintech, USA), Gli1 (ab49314; Abcam, Cambridge, MA, USA), Gli2 (18989-1-AP; Proteintech, USA), Gli3 (ab69838; Abcam, Cambridge, MA, USA), Ihh (ab39634; Abcam, Cambridge, MA, USA), Ptch1 (ab53715; Abcam, Cambridge, MA, USA), Sufu (#C54G2; Cell Signaling Technology, Beverly, MA, USA) and Smo (ab236465,Abcam, Cambridge, MA, USA). The β‐actin (BS6007M; Bioworld Technology, Bloomington, MN, USA) was used as the control for total protein.
Ab53715
Manufactured by Abcam
Sourced in United States, China
Ab53715 is a monoclonal antibody that specifically binds to the human CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Ab53281, by Abcam (9 mentions)
Ab49314, by Abcam (5 mentions)
Ab236465, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab134906, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab151796, by Abcam (3 mentions)
Ab217326, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Ab72130, by Abcam (2 mentions)
Ab52919, by Abcam (2 mentions)
Ab5694, by Abcam (2 mentions)
Anti gli1, by Abcam (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab39634, by Abcam (1 mentions)
24 protocols using ab53715
1
Bone Tissue Protein Immunoblotting
2
Western Blotting Analysis of Hedgehog Signaling
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Standard western blotting was conducted70 (link). Cell pellets were solubilized in RIPA buffer with 5% Halt Protease and Phosphatase Inhibitor Cocktail and 2% 0.5 mM EDTA (all Thermo Fisher Scientific). Extracts were centrifuged and the supernatant was then recovered for SDS-PAGE. Primary antibodies against calretinin (Santa Cruz, SC-365956, 1:500), PTCH1 (Abcam, ab53715, 1:1000), GLI1 (Abcam, ab134906, 1:500), GLI2 (Origene, TA804601, 1:500), Cyclin D1 (Cell Signaling, 92G2, #2978, 1:1000), FLAG M2 (Sigma, F1804, 1:1000), SUFU (Cell Signaling, #2522 s, 1:1000), and tubulin (Cell Signaling, DM1A, 1:1000). Conjugated secondary antibodies anti-rabbit (LI-COR, 926–68073, 1:10,000) and anti-mouse (LI-COR, 926–32212, 1:10,000) were used and detected using the Odyssey Infrared Imaging System (LI-COR Biosciences), as described (Supplementary Figs. 10 –13 )70 (link). Quantification and analysis were performed using ImageJ. Experiments were performed in triplicate.
3
Immunohistochemical Detection of Hedgehog Pathway
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Staining for SHH, IHH, PTCH1 and SMO was performed as follows: 4-μm sections from FFPE blocks were mounted onto microscope slides (Dako IHC Microscope Slides; Dako, Hamburg, Germany) and stored for 30 min in a humid chamber at 80 °C. Sections were dewaxed and heated in a target retrieval solution (EnVision FLEX; Agilent, Santa Clara, CA, USA) at either low pH (SHH, IHH) or high pH (SMO, PTCH) for 20 min. Blocking of nonspecific staining was accomplished with a commercial blocking agent (Dual Endogenous Enzyme Block S2003; Dako) for 5 min. For immunostaining, we used rabbit monoclonal antibodies against SHH (cat. no. ab53281), IHH (ab52919), SMO (ab72130) and PTCH1 (ab53715) (all Abcam Inc., Cambridge, MA, USA). The diluted antibodies were incubated at room temperature for either 30 min (SMO 1 : 500 and PTCH1 1 : 200) or 90 min (SHH 1 : 150 and IHH 1 : 250). The following steps of the immunohistochemistry procedures were performed in accordance with the manufacturer's recommendations for the system (Dako REAL Detection System K5005; Dako). Visualization of the antigens was achieved by a commercial stain (AP/Permanent Red; Abcam) with haematoxylin for counterstaining.
4
Protein Isolation and Western Blotting
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The protein isolation and western blotting were conducted according to the traditional protocol. Samples were probed with GNG7 (Abcam, USA; ab238868), SMO (Proteintech, China; 20,787-1-AP), GLI1 (Abcam, USA; ab134906), PTCH1 (Abcam, USA; ab53715) or GAPDH (Abcam, USA; ab9485) monoclonal antibody. Then, the samples were probed with Goat anti-mouse/anti-rabbit HRP antibodies. GAPDH was selected for internal controls.
5
Immunohistochemical Analysis of Carotid Arteries
6
Immunofluorescence Staining for Patched Protein
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Cells were seeded on coverslips in 24-well plates and grown to 80% confluence. Coverslips were washed twice with PBS, incubated 15 min with 4% paraformaldehyde (PFA), and then 20 min with PBS/Triton 0.1% to permeabilize cells. After blocking 30 min in PBS/2% BSA, slices were incubated at 4 °C overnight with rabbit anti-Patched antibody (Abcam ab53715; 1/1000) in PBS/0.1% BSA. After three washes in PBS/0.1% BSA, slides were incubated at room temperature during 1 h with a secondary anti-rabbit antibody coupled to Alexa 594 in PBS/0.1% BSA and washed three times before mounting using antifade reagent containing DAPI (SlowFade Gold Invitrogen, Villebon sur Yvette, France) to stain nuclei. Images were acquired with a Zeiss Axioplan 2 fluorescence microscope coupled to a digital charge-coupled device camera using a 40×/1.3 Plan NeoFluar objective and filters for Alexa 594.
7
Western Blot Analysis of Signaling Proteins
8
Immunohistochemical Analysis of Prostate Cancer
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Immunohistochemical staining was performed on TMA and formalin‐fixed, paraffin‐embedded tissues from PC‐3 cell line xenografts. Before staining, sections were dried for 1 hour at 60℃ and deparaffinized in xylene for 30 minutes, after which they were rehydrated by serial incubations in alcohol. Heat‐induced antigen retrieval was performed in citrate buffer at pH 6.0. Sections were subsequently blocked with goat serum and incubated with primary antibody [anti‐β2‐AR rabbit monoclonal antibody (ab182136; Abcam), anti‐Ki‐67 rabbit monoclonal antibody (9027; Cell Signaling), anti‐Shh rabbit monoclonal antibody (ab53281; Abcam), anti‐Gli1 rabbit polyclonal antibody (bs‐1206R; Bioss, China) or anti‐Ptch1 rabbit polyclonal antibody (ab53715; Abcam) overnight at 4°C (details were shown in Table 1 ), followed by Histostain‐SP (Streptavidin‐Peroxidase) kit reagent (SP0023; Beijing Biosynthesis Biotec, China), according to the manufacturer's instructions. The diaminobenzidine chromogen (ZSGB Biotec, China) was used for coloration. Slides were finally counterstained with hematoxylin. Negative controls were obtained after the omission of the primary antibody. Stained sections were examined and photographed on a bright‐field microscope (BX53; Olympus) connected to cellSens standard imaging software (Olympus).
9
Immunohistochemical Analysis of Ciliary and Signaling Proteins
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Freshly sampled human tissues, mouse tissues or cells were flushed with ice-cold PBS and fixed by incubation in 4% paraformaldehyde in PBS overnight at 4 °C. Fixed samples were dehydrated, embedded in paraffin, and sectioned. The sections were de-waxed and rehydrated. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Antigen retrieval was performed by boiling for 15 min in citrate buffer pH 6.0 or Tris-EDTA pH 9.0. Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology). Staining was performed using the Zsbio kit (Beijing, China) according to the manufacturer's instructions.
10
Western Blot Analysis of Hedgehog Pathway Proteins
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Nuclear and cytoplasmic proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit and quantified with a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturers’ instructions. PBMCs and total cell lysates were prepared in 1 × SDS buffer, directly analyzed by SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking in 5% BSA in Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with antibodies against Flag (F18804, Sigma, Inc.), HA (51064-2-AP, Proteintech), GLI3 (ab6050, Abcam), GLI1 (ab49314, Abcam), PTCH1 (ab53715, Abcam), SHH (ab32281, Abcam), GAPDH (ET1702-66, Huabio, Hangzhou, China), and β-actin (ET1701-80, Huabio). The antigen–antibody complexes were incubated with HRP-conjugated secondary antibodies (Bioworld, Nanjing, China) and visualized by an infrared imaging technique (Bio-Rad Laboratories, Inc.). The intensity of protein bands was quantified using ImageJ2 to calculate the ratios of IntDen (GLI3)/IntDen (β-Actin) to ensure that the detection of protein bands was linearized. The Student’s t-test was used for the statistical comparison of GLI3 protein relative quantification between patients and normal controls.
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