Comprehensive Immunoprecipitation-HPLC Analysis

Protein extracts (100 μg) were subjected to immunoprecipitation using a protein A/G agarose column (Amicogen, Korea). Protein A/G agarose columns were separately pre-incubated with 1 μg of 216 different antisera for; growth and proliferation-related proteins (n = 10), cMyc/MAX/MAD signaling proteins (n = 3), p53/Rb/E2F signaling proteins (n = 5), epigenetic modification-related proteins (n = 6), protein translation-related proteins (n = 5), RAS signaling proteins (n = 17), growth factor-related proteins (n = 16), NFkB signaling proteins (n = 12), cellular protection-related proteins (n = 15), upregulated inflammatory proteins (n = 26), downregulated inflammatory proteins (n = 13), p53-mediated apoptosis-related proteins (n = 17), FAS-mediated apoptosis-related proteins (n = 8), angiogenesis-related proteins (n = 20), osteogenesis-related proteins (n = 12), antioxidant-related proteins (n = 8), oncogenic proteins (n = 15), and control housekeeping proteins (n = 3) (Table 2).Table 2

Antibodies used in the study.

Signaling proteins	No.	Antibodies
Cellular proliferation	10	Ki-67^, PCNA^, CDK4^, PLK4^, lamin A/C, MPM2^, p14^, p16^, p21^, p27^*
cMyc/MAX/MAD signaling	3	cMyc^, MAX^, MAD^*
p53/Rb/E2F signaling	5 (2)	p53, Rb-1^#, E2F-1^*, (p21, CDK4)
Wnt/β-catenin signaling	5	Wnt1^, β-catenin^, APC^, snail^, TCF-1^*
Epigenetic modification	6	DMAP1^, histone H1^, KDM4D^$, HDAC-10^$, MBD4^, DNMT1^
Protein translation signaling	5	DOHH^, DHS^, elF5A-1^$, elF5A-2^$, eIF2AK3^*
RAS signaling	17	NRAS^$, KRAS^$, STAT3^, SOS-1^, SOS-2^, RAF-B^, JAK2^$, JNK-1^, ERK1^, Rab^, p-ERK^$, AP-1^@, SP-1^@, SP-3^@, AMPK^@, (PKC^, p-PKC^@)
Growth factor signaling	16	FGF-1^, FGF-2^, HGF^, TGF-β1^#, TGF-β2, SMAD4^, PDGF-A^, IGF-1^, IGFIIR^, GH^, GHRH^, HER1^, HER2^, ERβ^, insulin^@, Met^*
NFkB signaling	12 (3)	NFkB^, IKK^, GADD45^, MDR, mTOR^@, p38^, p-p38^, NRF-2^, IL-1^, (ERK1^, p-ERK^*, AMPK)
Upregulated inflammatory proteins	26 (2)	IL-12^, CD3^, CD28^, CD31^, CD34^, CD40^, CD56^, CD68^, CD80^, CD99^, LL-37^, M-CSF^, MMP-1^$, -2^$, -3^$, -9^$, -10^$, -12^$, TIMP-1^&, TIMP-2^&, CXCR4^, COX1^, COX2^, integrin- α5^, (TGF- β1^#, TGF- β2^*)
Downregulated inflammatory proteins	13 (1)	TNFα^@, IL-1^, IL-6^, IL-8^, IL-10^, IL-28^, LTA4H^&, α1- antitrypsin ^&, lysozyme^, CD20^$, cathepsin C^, cathepsin G^, cathepsin K^*,
Cellular protection-related	15 (2)	LC3, PLC- β2, PI3K, PKC^, p-PKC^, FAK^, caveolin-1^, PGC-1α^, HSP-27^, HSP-70^, HSP-90^, TGase 2^$, p63^$, (pAKT1/2/3^*, HO-1)
Antioxidant-related	8 (3)	HO-1^, SOD-1^, GST-1^, SVCT2^&, NOS-1^$, (PGC-1α ^$, LC3^, NRF-2)
p53-mediated cellular apoptosis	17 (1)	(p53^), PUMA^, NOXA^, MDM2^, BCL2^, BAX^, BAD^, BAK^, BID^, AIF^, APAF-1^, caspase 9^, c-caspase 9^, caspase 3^, c-caspase 3^, PARP^, c-PARP^*
FAS-mediated cellular apoptosis	8 (3)	FASL^, FAS^, FADD^, FLIP^, caspase 8^, (BID^, caspase 3^, c-caspase 3^)
Oncogenic proteins	15 (2)	PTEN^&, MUC1, MUC4, maspin^, BRCA1^&, BRCA2^&, NF-1^, ATM^, CEA^$, 14-3-3^, survivin^@, DMBT1^, TERT^, (pAKT1/2/3^*, MBD4)
Angiogenesis-related proteins	20 (7)	HIF^&, VEGF-A^, VEGF-C^, angiogenin^$, LYVE-1^, CMG2^$, vWF^$, FLT-4^$, ET-1^, PAI-1^, VEGFR^00, plasminogen^, leptin^, (CD31, MMP-2, MMP-9, MMP-10, FGF-1, FGF-2, PDGF-A)
Osteogenesis-related proteins	12 (2)	OPG^, RANKL^, BMP-2^, BMP-4^, ALP^, osteocalcin^, osteopontin^, osteonectin^, RUNX2^, osterix^, (HSP-90, cathepsin K)
Control housekeeping proteins	3	α-tubulin^, β-actin^, GAPDH^*
Total	216 (28)

*Santa Cruz Biotechnology, USA; ^#DAKO, Denmark; ^$Neomarkers, CA, USA; ^@ZYMED, CA, USA; ^&Abcam, Cambridge, UK; the number of antibodies overlapped; ().

Abbreviations: AMPK; AMP-activated protein kinase, pAKT; v-akt murine thymoma viral oncogene homolog, p-Akt1/2/3 phosphorylated (p-Akt, Thr 308), APAF-1; apoptotic protease-activating factor 1, AP-1; activating protein-1, BAD; BCL2 associated death promoter, BAK; BCL2 antagonist/killer, BAX; BCL2 associated X, BCL-2; B-cell leukemia/lymphoma-2, BID; BH3 interacting-domain death agonist, c-caspase 3; cleaved-caspase 3, CD3; cluster of differentiation 3, CDK4; cyclin dependent kinase 4, CEA; carcinoembryonic antigen, CMG2: capillary morphogenesis protein 2, COX-1; cyclooxygenase-2, COX-2; cyclooxygenase-2, c-PARP; cleaved- PARP (poly-ADP ribose polymerase), DMAP1; DNA methyltransferase 1 associated protein, DMBT1; deleted in malignant brain tumors 1, DOHH; deoxyhypusine hydroxylase, DHS; deoxyhypusine synthase, E2F-1; transcription factor, eIF2AK3 (PERK); eukaryotic translation initiation factor 2 (protein kinase R (PKR)-like endoplasmic reticulum kinase), elF5A-1; eukaryotic translation initiation factor 5A-1, elF5A-2; eukaryotic translation initiation factor 5A-2, ERβ; estrogen receptor beta, ERK; extracellular signal-regulated protein kinases, ET-1: endothelin-1, FAS; CD95/Apo1, FASL; FAS ligand, FADD; FAS associated via death domain, FGF-1; fibroblast growth factor-1, FLIP; FLICE-like inhibitory protein, FLT-4; Fms-related tyrosine kinase 4, GADD45; growth arrest and DNA-damage-inducible 45, GAPDH; glyceraldehyde 3-phosphate dehydrogenase, GH; growth hormone, GHRH; growth hormone-releasing hormone, GST-1; glutathione S-transferase ω 1, HDAC-10; histone deacetylase 10, HIF-1α: hypoxia inducible factor-1α, HO-1; heme oxygenase 1, HER1; human epidermal growth factor receptor 1, HGFα; hepatocyte growth factor α, HSP-70; heat shock protein-70, IKK; ikappaB kinase, IGF-1; insulin-like growth factor 1, IGFIIR; insulin-like growth factor 2 receptor, IgK; immunoglobulin kappa (light chain), IL-1; interleukin-1, KDM4D; Lysine-specific demethylase 4D, JNK-1; Jun N-terminal protein kinase, KRAS; V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, LC3; microtubule-associated protein 1 A/1B-light chain 3, LYVE-1: lymphatic vessel endothelial hyaluronan receptor 1, MAX; myc-associated factor X, MBD4; methyl-CpG-binding domain protein 4, M-CSF; macrophage colony-stimulating factor, MDM2; mouse double minute 2 homolog, MDR; multiple drug resistance, MMP-1; matrix metalloprotease-1, MPM2; mitotic protein monoclonal 2, mTOR; mammalian target of rapamycin, cMyc; V-myc myelocytomatosis viral oncogene homolog, NFkB; nuclear factor kappa-light-chain-enhancer of activated B cells, NOS-1; nitric oxide synthase 1, NRAS; neuroblastoma RAS Viral Oncogene homolog, NRF2; nuclear factor (erythroid-derived)-like 2, p14, p16, p21, p27, p38, PAI-1; plasminogen activator inhibitor-1, PARP; poly-ADP ribose polymerase, PCNA; proliferating cell nuclear antigen, PDGF-A: platelet-derived growth factor-A, PLC-β2; 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterse β-2, PI3K; phosphatidylinositol-3-kinase, PLK4; polo like kinase 4 or serine/threonine-protein kinase, PKC; protein kinase C, p-p38; phosphor-p38, PTEN; phosphatase and tensin homolog, RANKL; receptor activator of nuclear factor kappa-B ligand, Rb-1; retinoblastoma-1, RUNX2; Runt-related transcription factor-2, SMAD4; mothers against decapentaplegic, drosophila homolog 4, SOD-1; superoxide dismutase-1, SP-1; specificity protein 1, STAT3; signal transducer and activator of transcription-3, TGF-β1; transforming growth factor-β1, TERT; human telomerase reverse transcriptase, TNFα; tumor necrosis factor-α, β-actin, 14-3-3, VEGF vascular endothelial growth factor, VEGFR2: vascular endothelial growth factor receptor 2, p-VEGFR2: vascular endothelial growth factor receptor 2 (Y951), vWF: von Willebrand factor.

Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40) and incubated in protein A/G agarose (Amicogen, Korea) columns at 4 °C for 1 hour (columns were placed on a rotating stirrer during incubation). After washing each column with sufficient phosphate buffered saline solution, target proteins were eluted with 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using a HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Korea). Elution was performed using 0.15 M NaCl/20% acetonitrile solution at 0.4 mL/min for 30 min, and proteins were detected by UV spectroscopy at 280 nm. Control and experimental samples were run sequentially to allow comparisons^{12 (link),30 ,31}. For IP-HPLC, whole protein peak areas (mAU*s) were calculated after subtracting negative control antibody peak areas, and the square roots of protein peak areas were calculated to normalize concentrations (Supplementary Fig. 2). Protein percentages in total proteins in experimental and control groups were plotted. Analyses were repeated two to six times to achieve mean standard deviations of ≤±5%. Results were analyzed using the Chi-squared test.
The expressions of housekeeping proteins, that is, β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. Expressional changes of housekeeping proteins were adjusted to <±5% using a proportional basal line algorithm. To describe protein expressional changes, we tentatively defined a ≤±5% change as minimal, ±5–10% as slight, ±10–20% as meaningful, and a ≥±20% change as marked.

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Kim M.K., Yoon C.S., Kim S.G., Park Y.W., Lee S.S, & Lee S.K. (2019). Effects of 4-Hexylresorcinol on Protein Expressions in RAW 264.7 Cells as Determined by Immunoprecipitation High Performance Liquid Chromatography. Scientific Reports, 9, 3379.

Publication 2019

Corresponding Organization :

Other organizations : Gangneung–Wonju National University

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Variable analysis

independent variables

Protein extracts (100 μg) subjected to immunoprecipitation using a protein A/G agarose column

dependent variables

Expressions of 216 different proteins measured by IP-HPLC

control variables

Protein A/G agarose columns pre-incubated with 1 μg of 216 different antisera
Control antibody peak areas subtracted from experimental peak areas
Expressions of housekeeping proteins (β-actin, α-tubulin, GAPDH) used as internal controls

controls

Negative control antibody used
Control and experimental samples run sequentially for comparison

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