ab12337, by Abcam - Product details

1

Immunofluorescent Labeling of Proteins

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The immunofluorescent labelling of proteins was conducted as described previously (Jamsai et al. 2011 ). The well-established primary antibodies (HENMT1 (specificity proven on knockout in (Lim et al. 2015) ) and PIWIL1 (Abcam ab12337); both 5 µg/mL) were incubated at 4°C overnight. Secondary antibodies anti-goat AF 455 and anti-rabbit 455 (Life Technologies; 4 µg/mL) were incubated 1 h at room temperature in the darkness. Sections were counterstained with DAPI and coverslipped with fluorescence mounting media (DAKO). Pictures were taken on a Leica SP8 confocal microscope (Leica).
For immunochemistry sections were labelled as described previously (Fietz et al. 2014 (link)) using the PIWIL1 antibody (Abcam ab12337; 7 µg/mL). For a negative control, the primary antibody was pre-incubated with an excess of the immunizing peptide ab13827 (Abcam). Pictures were taken with a Leica ICC50 HD Microscope Camera on a Leica DM750 light microscope (Leica).

Hempfling A.L., Lim S.L., Adelson D.L., Evans J., O'Connor A.E., Qu Z.P., Kliesch S., Weidner W., O'Bryan M.K, & Bergmann M. (2017). Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression. Reproduction (Cambridge, England), 154(4).

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2

Immunohistochemical Localization of A-MYB and MIWI in Human Testis

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Paraffin embedded human testis tissue fixed with 4% (v/v) formaldehyde were obtained from the UMass Medical School Tissue and Tumor Bank or Kyle Orwig. Embedded tissues were sectioned at 4 μm thickness and stained with hematoxylin and eosin (H&E) by the UMass Medical School Morphology Core Facility.
Immunohistochemical (IHC) staining was performed using standard protocols. Briefly, testis sections were de-paraffinized with xylene, dehydrated with ethanol, and heated with 1 mM citrate buffer (pH 6.0) to retrieve antigen. Endogenous peroxidase activity was inactivated with 3% (w/v) hydrogen peroxide for 10 min at room temperature. Tissues were blocked with 5% (v/v) horse serum using ImmPRESS HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection Kit (Vector labs; MP-7401). Sections were then incubated with rabbit anti-A-MYB (Sigma, HPA008791; 1:400 dilution) or rabbit anti-MIWI (Abcam, ab12337; 1:400 dilution) antibody overnight at 4°C. Secondary HRP anti-rabbit antibody (Vector labs; MP-7401) was applied for 1 h at room temperature, followed by incubation with substrate/chromogen (Fisher Scientific, TA-125-QHDX). Finally, slides were counterstained with hematoxylin, dehydrated and sealed with a coverslip. H&E and IHC images were captured using a Leica DMi8 microscope.

Özata D.M., Yu T., Mou H., Gainetdinov I., Colpan C., Cecchini K., Kaymaz Y., Wu P.H., Fan K., Kucukural A., Weng Z, & Zamore P.D. (2019). Evolutionarily Conserved Pachytene piRNA Loci are Highly Divergent among Modern Humans. Nature ecology & evolution, 4(1), 156-168.

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3

Immunohistochemical Localization of A-MYB and MIWI in Human Testis

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Paraffin embedded human testis tissue fixed with 4% (v/v) formaldehyde were obtained from the UMass Medical School Tissue and Tumor Bank or Kyle Orwig. Embedded tissues were sectioned at 4 μm thickness and stained with hematoxylin and eosin (H&E) by the UMass Medical School Morphology Core Facility.
Immunohistochemical (IHC) staining was performed using standard protocols. Briefly, testis sections were de-paraffinized with xylene, dehydrated with ethanol, and heated with 1 mM citrate buffer (pH 6.0) to retrieve antigen. Endogenous peroxidase activity was inactivated with 3% (w/v) hydrogen peroxide for 10 min at room temperature. Tissues were blocked with 5% (v/v) horse serum using ImmPRESS HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection Kit (Vector labs; MP-7401). Sections were then incubated with rabbit anti-A-MYB (Sigma, HPA008791; 1:400 dilution) or rabbit anti-MIWI (Abcam, ab12337; 1:400 dilution) antibody overnight at 4°C. Secondary HRP anti-rabbit antibody (Vector labs; MP-7401) was applied for 1 h at room temperature, followed by incubation with substrate/chromogen (Fisher Scientific, TA-125-QHDX). Finally, slides were counterstained with hematoxylin, dehydrated and sealed with a coverslip. H&E and IHC images were captured using a Leica DMi8 microscope.

Özata D.M., Yu T., Mou H., Gainetdinov I., Colpan C., Cecchini K., Kaymaz Y., Wu P.H., Fan K., Kucukural A., Weng Z, & Zamore P.D. (2019). Evolutionarily Conserved Pachytene piRNA Loci are Highly Divergent among Modern Humans. Nature ecology & evolution, 4(1), 156-168.

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4

Immunoprecipitation of piRNA-Binding Proteins

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Mouse testes were homogenized in lysis buffer (20 mM HEPES, pH 7.3, 150 mM NaCl, 2.5 mM MgCl₂, 0.2 % NP-40, and 1 mM DTT) with protease inhibitor cocktail (Thermo Scientific) and RNase inhibitor (Promega). The lysates were centrifuged at 13,000×g for 10 min after sonication. The supernatants were collected and pre-cleared with protein-A agarose beads (Roche) at 4 °C for 2 h. Anti-MILI (PM044, MBL) or anti-MIWI (ab12337, Abcam) antibodies were used for immunoprecipitation and protein-A agarose beads were added to the lysates and incubated for 4 h to capture immunocomplexes. The beads were then collected and washed in lysis buffer for five times. Immunoprecipited piRNAs were isolated using Trizol reagent (Thermo Scientific) for downstream piRNA labeling and small RNA library construction experiments.

Ding D., Liu J., Midic U., Wu Y., Dong K., Melnick A., Latham K.E, & Chen C. (2018). TDRD5 binds piRNA precursors and selectively enhances pachytene piRNA processing in mice. Nature Communications, 9, 127.

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5

Immunoblotting Analysis of Autophagy Markers

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The following primary antibodies were used in this study: anti-PIWIL1 antibody (ab12337; Abcam, Cambridge, UK), anti-p-mTOR antibody (ab109268; Abcam), anti-LC3 I/II (#2775S; Cell Signaling Technology, Danvers, MA, USA), anti-P62/SQSTM1 (#8025S; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-phosphor-NAK/TBK (ab109272; Abcam), anti-NAK/TBK (A5497; Bimake), anti-optineurin (ab213556; Abcam), anti-β-actin (#4970; Cell Signaling Technology), anti-Akt (#4691; Cell Signaling Technology), anti-phospho-Akt (Ser473) (#9271; Cell Signaling Technology), anti-Ki67 (#4685; Cell Signaling Technology), anti-Bcl-2 (A5010; Bimake), anti-OCT4 (60242-1-Ig; Proteintech), and anti-Nanog (#4903T; Cell Signaling Technology). Goat anti-mouse and goat anti-rabbit antibodies (Biosciences) were used as the secondary antibodies for western blotting. Dexamethasone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), and bortezomib was purchased from Selleck Chemicals. 3-Methyladenine (3-MA) and the mitophagy inhibitor cyclosporin A (CsA) were purchased from MedChemExpress (NJ, USA). All were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C for up to 6 months. For all cell-based experiments, drugs were diluted at least by 1:1000 to ensure that the final DMSO concentration was lower than 0.1%.

Wang Y., Yao L., Teng Y., Yin H, & Wu Q. (2022). PIWIL1 Drives Chemoresistance in Multiple Myeloma by Modulating Mitophagy and the Myeloma Stem Cell Population. Frontiers in Oncology, 11, 783583.

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6

Immunofluorescence Assay for PIWIL1 Detection

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For IF, cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS-Tween; permeabilization was performed in 0.1% Triton-X-100 in PBS for 10 min, then cells were incubated in blocking buffer (0.5% BSA in PBS-Tween) for 1 h. Slides were incubated at 4 °C overnight with rabbit anti-PIWIL1 (ab12337, Abcam, Cambridge, UK). The next day, slides were washed with 0.5% BSA-PBS-Tween and incubated for 45 min at room temperature with Alexa Fluor 488 goat anti-rabbit IgG (Thermo-Fisher, Carlsbad, CA) secondary antibody in PBS. For nuclei staining, 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, St. Louis, MS, USA) was used. Images were collected with a Leica DM6000 B confocal microscope and processed with ImageJ software (https://imagej.net).

Sellitto A., Geles K., D’Agostino Y., Conte M., Alexandrova E., Rocco D., Nassa G., Giurato G., Tarallo R., Weisz A, & Rizzo F. (2019). Molecular and Functional Characterization of the Somatic PIWIL1/piRNA Pathway in Colorectal Cancer Cells. Cells, 8(11), 1390.

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7

Immunoprecipitation of MILI and MIWI Proteins

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Mouse testes were collected and homogenized using lysis buffer (20 mM HEPES pH 7.3, 150 mM NaCl, 2.5 mM MgCl₂, 0.2% NP-40, and 1 mM DTT) with protease inhibitor cocktail (Thermo Scientific) and RNase inhibitor (Promega). After sonication, testis lysates were centrifuged at 12,000 rpm for 10 min. The supernatants were pre-cleared using protein-A agarose beads (Roche) for 2 h. Anti-MILI (1:500; PM044, MBL) or anti-MIWI (1:100; ab12337, Abcam) antibodies together with protein-A agarose beads were added to the lysates and incubated for 4 h. The beads were washed in lysis buffer for five times. Immunoprecipitated RNAs were isolated from the beads using Trizol reagent (Thermo Scientific) for piRNA labeling or small RNA library construction. For protein detection, immunoprecipitated beads were boiled in protein loading buffer for 5 min. Western blotting of MILI or MIWI was performed as described above.

Ding D., Liu J., Dong K., Midic U., Hess R.A., Xie H., Demireva E.Y, & Chen C. (2017). PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice. Nature Communications, 8, 819.

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8

Profiling piRNA-Piwi Interactions

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The antibody used to immunoprecipitate (IP) piRNAs is anti-PIWIL1 (Abcam, ab12337), originally raised against the human PIWIL1 protein C-terminal peptide. Immunoprecipitation, Illumina library preparation, deep sequencing, and bioinformatic analysis of Piwi-associated small RNAs from the sexual progeny of both WT and backcrossed cells followed our published protocol.¹⁹ (link) Small RNA sequences were mapped onto the micronuclear region containing contig11396-TEBPβ with the gmapper command in the SHRiMP software package,⁶² (link) setting the threshold score to 80% of the maximum possible score using the ‘-h 80%' flag. To ensure comparable depth between control and fusion libraries, we subsampled to equivalent sequencing depth of 35 million reads per library.

Bracht J.R., Wang X., Shetty K., Chen X., Uttarotai G.J., Callihan E.C., McCloud S.S., Clay D.M., Wang J., Nowacki M, & Landweber L.F. (2016). Chromosome fusions triggered by noncoding RNA. RNA Biology, 14(5), 620-631.

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9

Quantifying PIWIL Protein Levels

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Protein was extracted from the homogenized tissues using RIPA buffer and estimated using BCA Protein Assay Kit (Thermo Fisher Scientific). The expression of PIWIL protein was evaluated by sodium dodecyl sulphate (SDS) acrylamide gel electrophoresis and immunoblotting of total protein extracts using rabbit anti-PIWIL1 (ab12337, Abcam), rabbit anti-PIWIL2 (ab26408, Abcam), rabbit anti-PIWIL4 (ab111714, Abcam), mouse anti β-actin (ab6276, Abcam) and goat anti-rabbit IgG H&L (HRP) (ab97051, Abcam).

Singh G., Roy J., Rout P, & Mallick B. (2018). Genome-wide profiling of the PIWI-interacting RNA-mRNA regulatory networks in epithelial ovarian cancers. PLoS ONE, 13(1), e0190485.

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10

PIWIL1 Overexpression in HCT 116 Cells

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For the transient overexpression of PIWIL1, HCT 116 cells were transfected with the human full-length cDNA clone pCMV6-PIWIL1 (RC205269) or with control pCMV6-Entry mammalian vector (PS100001) (Origene, Herford, Germany). At 24 h prior to transfection, HCT 116 cells at the exponential growth phase were seeded in 100 mm culture dishes; the next day, plates at 60% confluency were washed and re-fed with culture medium shortly before transfection. A total of 15 µg of DNA was mixed with linear 25 kDa polyethyleneimine (PEI) (Polysciences, Eppenheim, Germany) and incubated for 20 min at room temperature, then the DNA/PEI mixture was added to plates. After 24 h from transfection, HCT 116-pCMV6-PIWIL1 cells were analyzed by immunofluorescence using rabbit anti-PIWIL1 (ab12337, Abcam, Cambridge, UK); fluorescence images were collected with a LEICA DM6000 B Confocal Microscope and used to evaluate and quantify transfection efficiency, which was found to be ~20%.

Sellitto A., Geles K., D’Agostino Y., Conte M., Alexandrova E., Rocco D., Nassa G., Giurato G., Tarallo R., Weisz A, & Rizzo F. (2019). Molecular and Functional Characterization of the Somatic PIWIL1/piRNA Pathway in Colorectal Cancer Cells. Cells, 8(11), 1390.

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Ab12337

Lab products found in correlation

13 protocols using ab12337

Immunofluorescent Labeling of Proteins

Immunohistochemical Localization of A-MYB and MIWI in Human Testis

Immunohistochemical Localization of A-MYB and MIWI in Human Testis

Immunoprecipitation of piRNA-Binding Proteins

Immunoblotting Analysis of Autophagy Markers

Immunofluorescence Assay for PIWIL1 Detection

Immunoprecipitation of MILI and MIWI Proteins

Profiling piRNA-Piwi Interactions

Quantifying PIWIL Protein Levels

PIWIL1 Overexpression in HCT 116 Cells

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