Ha c29f4

Manufactured by Cell Signaling Technology

Sourced in United States

HA (C29F4) is a monoclonal antibody that recognizes the hemagglutinin (HA) epitope tag. It is a laboratory reagent used for the detection and purification of HA-tagged proteins.

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Lab products found in correlation

Flag m2, by Merck Group (2 mentions) Flag tag (flag), by Thermo Fisher Scientific (2 mentions) C myc, by Thermo Fisher Scientific (2 mentions) Streptavidin irdye800, by LI COR (2 mentions) Goat anti rabbit dylight 800, by Thermo Fisher Scientific (2 mentions) Irdye 800cw goat anti mouse, by LI COR (2 mentions) α tubulin 2144, by Cell Signaling Technology (2 mentions) Perk sc 7383 and sc 16982 r, by Santa Cruz Biotechnology (2 mentions) Etv5 wh0002119m2, by Merck Group (2 mentions) Gfp sc 9996, by Santa Cruz Biotechnology (2 mentions) Vinculin v9264, by Merck Group (2 mentions) Polyclonal erk m5670, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Myc 9e10, by Santa Cruz Biotechnology (1 mentions) Rm4 5, by Thermo Fisher Scientific (1 mentions) β actin ac 15, by Merck Group (1 mentions) Ra3 6b2, by Thermo Fisher Scientific (1 mentions) Ha f 7, by Santa Cruz Biotechnology (1 mentions) Gst b 14, by Santa Cruz Biotechnology (1 mentions) P53 do 1, by Santa Cruz Biotechnology (1 mentions)

11 protocols using ha c29f4

Western Blot and Flow Cytometry Analysis

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The following Abs were used for Western blot analyses or flow cytometry analysis: p53 (DO-1, Santa Cruz), p21 (DSC60, Cell Signaling Technology, Danvers, MA), β-actin (AC-15, Sigma-Aldrich, St. Louis), His (Invitrogen), GST (B-14, Santa Cruz), HA (F-7, Santa Cruz), HA (C29F4, Cell Signaling Technology), Myc (9E10, Santa Cruz), Ab against mouse DD1α (MH5A, BioLegend), Ab against human PD-1 (J116, eBioscience), Ab against human PD-L1 (M1H1, eBioscience), CTLA4 (C-19, Santa Cruz), ICOSL (LifeSpan BioSciences), Ab against human CD14 APC (61D3, eBioscience), Ab against mouse F4/80 APC (BM8, eBioscience), Ab against mouse CD45R/B220 (RA3–6B2, eBioscience), Ab against human CD4-APC (OKT4, eBioscience), Ab against mouse CD4-APC (RM4–5, eBioscience), Ab against human CD8-APC (SK1, eBioscience) and Ab against mouse CD8a⁺-APC (53–6.7, eBioscience). Rabbit polyclonal and mouse monoclonal Ab against DD1α was raised against human DD1α (amino acids 33–311) as an immunogen. Beads immobilized with Abs against HA (Roche) or Myc (9B11, Cell Signaling) were used for immunoprecipitations. Recombinant human PD-1–Ig, mouse PD-1–Ig, human PD-L1–Ig, mouse PD-L1–Ig, and control Ig proteins were from R&D Systems.

Yoon K.W., Byun S., Kwon E., Hwang S.Y., Chu K., Hiraki M., Jo S.H., Weins A., Hakroush S., Cebulla A., Sykes D.B., Greka A., Mundel P., Fisher D.E., Mandinova A, & Lee S.W. (2015). Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53. Science (New York, N.Y.), 349(6247), 1261669.

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Antibody Dilution Protocol for Immunoblotting

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Antibodies purchased from Sigma‐Aldrich, with dilutions for immunoblotting, were as follows: F3165, monoclonal anti‐FLAG 1:10,000; T5168, monoclonal anti‐α tubulin 1:8,000; A0168 HRP (horseradish peroxidase)‐conjugated anti‐mouse IgG 1:10,000, A0545 HRP‐conjugated anti‐rabbit IgG 1:10,000; HPA036582, polyclonal anti‐FAM35A 1:200. D153‐8 agarose‐conjugated anti‐GFP (RQ2) was purchased from MBL International Corporation. From Clontech, polyclonal anti‐GFP antibody 632592 was used at 1:200 (for immunostaining); monoclonal anti‐GFP 632381, 1:3,000. Anti‐RAD51 antibody (B01P, 1:2,000) was purchased from Abnova. Goat anti‐mouse IgG (H+L) secondary antibody, Alexa Fluor^® 594 conjugate (A‐11005) 1:2,000, goat anti‐rabbit IgG (H+L) secondary antibody, Alexa Fluor^® 488 conjugate (A‐11008) 1:2,000, and goat anti‐mouse IgG (H+L) secondary antibody, Alexa Fluor^® 488 conjugate (A‐11001) 1:2,000 were purchased from Thermo Fisher. Anti‐FANCD2 (EPR2302, 1:2,000 dilution) was purchased from GeneTex, Inc. From Cell Signaling Technology, monoclonal anti‐HA (C29F4) was used at 1:1,000.

Tomida J., Takata K., Bhetawal S., Person M.D., Chao H., Tang D.G, & Wood R.D. (2018). FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1‐defective cells. The EMBO Journal, 37(12), e99543.

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Glioblastoma Cell Lines and Astroglia Culture

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GBM cell lines (U-118 MG, A172, U-87 MG, U-251 MG, and LN-229) and normal astroglia cells (SVGP12) were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S). The 293FT cell line was culture as previously described [2 (link)]. All cell lines were tested mycoplasma-negative.
The FBS, antibiotics, and DMEM media were obtained from Thermo Fisher Scientific (MA, USA). Crystal violet and Dimethyl sulfoxide (DMSO) were purchased from Sigma (MO, USA). The RANBP10 (21107), E-cadherin (20874), p21 (10355), CyclinD1 (60186), Myc (60003), and Tubulin (11224) antibodies were obtained from Proteinch (Wuhan, China). The c-Myc (32072) and FBXW7 (109617) were obtained from Abcam (Shanghai, China); The CDK4 (12790), CDK6 (13331), N-cadherin (13116), Flag (14793), and HA (C29F4) antibodies were obtained from Cell Signaling Technology (MA, USA).

Hou J., Liu Y., Huang P., Wang Y., Pei D., Tan R., Zhang Y, & Cui H. (2021). RANBP10 promotes glioblastoma progression by regulating the FBXW7/c-Myc pathway. Cell Death & Disease, 12(11), 967.

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Immunoprecipitation and Western Blotting Analysis

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Immunoprecipitation and Western blotting analyses were performed as we previously described (23 (link)). Briefly, cells were lysed, centrifuged, and then protein concentrations were determined. Equal amounts of cell lysates were immunoprecipitated with specific antibodies and protein G-agarose beads (Invitrogen). Standard Western blotting was done with antibody against β-actin (I-19), STAT1 (C-111), STAT5 (G-2), and STAT3 (H-190) (Santa Cruz Biotechnology); Flag (M2, Sigma-Aldrich); HA (#66006-1-Ig), Lamin B1 (Proteintech Group); TRIM59 (ab69639, Abcam); EGFR (D38B1), phospho-STAT3 (Y705) (D3A7), phospho-EGFR (Y1173) (53A5), STAT3 (124H6), TCPTP (TC45)(D7T7D), Myc (9B11), Tubulin (#2148), and HA (C29F4) (Cell Signaling Technology).

Sang Y., Li Y., Song L., Alvarez A.A., Zhang W., Lv D., Tang J., Liu F., Chang Z., Hatakeyama S., Hu B., Cheng S.Y, & Feng H. (2018). TRIM59 promotes gliomagenesis by inhibiting TC45 dephosphorylation of STAT3. Cancer research, 78(7), 1792-1804.

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Western Blot Antibody Immunodetection

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The following primary and secondary antibodies were used: Gα_i1/2 (anti-sera) ^{58 (link)}, c-Myc (13–2500, Invitrogen), GFP (A11122, Invitrogen), HA (C29F4, Cell Signaling), FLAG (PA1–984B, Invitrogen). Streptavidin-IRDye800 was from LI-COR (925–32230). Primary antibodies were diluted in 3% bovine serum albumin (BSA) and 0.1% sodium azide and incubated with blots overnight at 4°C. Streptavidin-IRDye800 was incubated for 1 hour at room temperature. For secondary antibodies, goat anti-rabbit DyLight 800 (SA535571, Invitrogen) and goat anti-mouse IRDye 800CW (926–32210, LI-COR) were used at 1:10,000.

Lefevre T.J., Wei W., Mukhaleva E., Venkata S.P., Chandan N.R., Abraham S., Li Y., Dessauer C.W., Vaidehi N, & Smrcka A.V. (2023). Stabilization of Interdomain Interactions in G protein αi Subunits Determines Gαi Subtype Signaling Specificity. bioRxiv.

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Immunoprecipitation and Western Blotting

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For immunoprecipitation, 30 μg of total protein from transfected HEK 293 cells was incubated with HA (12CA5, Roche) or Flag (M2, Sigma) antibodies in 50 μL of IP buffer at 4 °C for 4 h. Protein G Mag sepharose (Dynabeads, Invitrogen) were added to the mixture and incubated overnight. The complex was placed on a magnet and washed three times. Proteins eluted from the sepharose beads were subjected to SDS-PAGE and immunoblotting using HA (C29F4, Cell Signaling) or Flag (2EL-1B11, Merck Millipore) antibodies. For sequential reprobing of the same blots, the membranes were stripped and hybridized with another primary antibody. Blots were developed using an enhanced chemiluminescence detection kit (Amersham) and protein intensities were quantified using Image J software (version 1.48, http://imagej.nih.gov/ij/).

Lee C.P., Chiang S.L., Ko A.M., Liu Y.F., Ma C., Lu C.Y., Huang C.M., Chang J.G., Kuo T.M., Chen C.L., Tsai E.M, & Ko Y.C. (2016). ALPK1 phosphorylates myosin IIA modulating TNF-α trafficking in gout flares. Scientific Reports, 6, 25740.

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Quantitative Western Blotting Analysis

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The following primary and secondary antibodies were used: Gα_i1/2 (anti-sera) (56 (link)), c-Myc (13-2500, Invitrogen), GFP (A11122, Invitrogen), HA (C29F4, Cell Signaling Technology), FLAG (PA1-984B, Invitrogen). Streptavidin-IRDye800 was from LI-COR (925-32230). Primary antibodies were diluted in 3% bovine serum albumin and 0.1% sodium azide and incubated with blots overnight at 4 °C. Streptavidin-IRDye800 was incubated for 1 h at room temperature (RT). For secondary antibodies, goat anti-rabbit DyLight 800 (SA535571, Invitrogen) and goat anti-mouse IRDye 800CW (926-32210, LI-COR) were used at 1:10,000.

Lefevre T.J., Wei W., Mukhaleva E., Meda Venkata S.P., Chandan N.R., Abraham S., Li Y., Dessauer C.W., Vaidehi N, & Smrcka A.V. (2024). Stabilization of interdomain interactions in G protein α subunits as a determinant of Gαi subtype signaling specificity. The Journal of Biological Chemistry, 300(5), 107211.

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Antibody Characterization for Epigenetic Studies

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Antibodies used in this study were DICER (3363, rabbit; Cell Signaling), ZRF1 (NBP2-12802; Novus Biologicals). RING1B clone (D22F2, 5694, rabbit; Cell Signaling), XPA (GTX103168, mouse; Genetex), XPA (FL-273, rabbit; Santa Cruz) H2B (V119 8135, mouse; Cell Signaling), FLAG (mouse; Sigma), H4K20me2 (39173, rabbit; Active Motif), H4K16ac (39930, rabbit; Active Motif), H3K36me2 (39256, rabbit; Active Motif), CPD (mouse; CosmoBio), HA (C29F4, rabbit; Cell Signaling), XPB (S-19, rabbit; Santa Cruz), MMSET (39880, mouse; Active Motif), 53BP1 (NB100-304, rabbit; Novus), and SETD8 (C18B7, rabbit; Cell Signaling).

Chitale S, & Richly H. (2018). DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites. The Journal of Cell Biology, 217(2), 527-540.

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Western Blot Analysis of Cellular Protein Markers

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RIPA buffer (Beyotime, Jiangsu, China) with protease inhibitor and phosphatase inhibitor cocktails (Beyotime) was used to extract protein lysates. Western blotting procedures were performed as described previously [19 (link)]. Antibodies against c-MYC (E5Q6W) (1:1000, #18,583), GAPDH (14C10) (1:1000, #2118), CCND1 (E3P5S) (1:1000, #55,506), c-Jun (60A8) (1:1000, #9165), AGO2 (C34C6) (1:1000, #2897), CDK4 (D9G3E) (1:1000, #12,790), caspase-3 (1:1000, #9662), cleaved caspase-3 (Asp175) (5A1E) (1:1000, #9664), PARP (46D11) (1:1000, #9532), ubiquitin (E6K4Y) (1:1000, #20,326) and HA (C29F4) (1:1000, #3724) were purchased from Cell Signaling Technology (CST, MA, USA). MYCBP (2E9) (1:1000, sc-517,020) was purchased from Santa Cruz (Cambridge, MA, USA). Secondary antibodies: anti-rabbit IgG, HRP-linked antibody (1:1000, #7074) and HRP-conjugated anti-mouse IgG, antibody (1:1000, #7076) were purchased from CST (USA).

Zhang Y., Zhan Y., Liu Z., Guo H., Liu D, & Chen C. (2024). Circ_0002669 promotes osteosarcoma tumorigenesis through directly binding to MYCBP and sponging miR-889-3p. Biology Direct, 19, 25.

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Antibody Source and Dilution Protocol

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The following antibodies were obtained from Cell Signaling Technologies: HA (C29F4) (1:6000), Lamin(A/C) (2032), pERK (4370), ubiquitin (3933), PJA2 (40180), and β-TrCP (D13F10), β-actin (8H10D10) (1:20,000), myc tag (71D10), and α-tubulin (2144) (1:5000). pERK (sc-7383 and sc-16982-R), PJA1 (sc-517068), and GFP (sc-9996) (1:6000) were obtained from Santa Cruz Biotechnology. Ki67 was obtained from Dako. Phospho Ser/Thr (ab17464), T7 tag (ab9138), capicua (ab123822), ETV1 (ab81086), Renilla (ab187338), and myc protein (ab3207) were obtained from Abcam. The following antibodies were purchased from Millipore T7 tag (69522), capicua (ABN446) and capicua (MABN449). FLAG-M2 (F1804), β-actin (A5316) (1:10,000), vinculin (V9264) (1:30,000), ETV5 (WH0002119M2), and polyclonal ERK (M5670) antibodies (1:5000) were obtained from Sigma. PJA1 (MBS153701) was purchased from MyBioSource.com. All antibodies were utilized at a 1:1,000 dilution unless otherwise specified.

Bunda S., Heir P., Metcalf J., Li A.S., Agnihotri S., Pusch S., Yasin M., Li M., Burrell K., Mansouri S., Singh O., Wilson M., Alamsahebpour A., Nejad R., Choi B., Kim D., von Deimling A., Zadeh G, & Aldape K. (2019). CIC protein instability contributes to tumorigenesis in glioblastoma. Nature Communications, 10, 661.

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