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8 protocols using anti β actin ac 15

1

Plasmid-Encoded Protein Expression Protocols

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Expression plasmids encoding CD63 and syntenin-1 were described previously50 (link)80 (link)81 (link). CD63/T7 was generated using a standard PCR protocol generating a CD63 protein which resembles the C-terminus of Tspan7 (primers are available upon request). CD63 was amplified from clone IRAUp969A121D6 (imaGenes, Berlin, Germany) by standard PCR. The CD63 sequence was then cloned into the XhoI-KpnI site of the pEGFP-C1 (Clontech, Heidelberg, Germany) vector. The ALIX-HA containing plasmid (pCAGGS-HA-AIP1) was a kind gift from Prof. Takemasa Sakaguchi (Hiroshima, Japan)82 (link). The ALIX V-domain containing plasmid (pGST2-ALIX364–716) was a kind gift from Prof James Hurley (NIH, USA). The HPV16 L1-specific antibodies mouse mAbs L1-7, H56.E, and 16L1-312F and rabbit pAb K75 (detecting HPV16, 18, and 31) have been described previously22 (link)24 (link)29 (link)47 (link)83 (link). The CD63 antibody 6H1 was described previously84 (link), the CD63 mAb sc-5275, rat CD63 mAb AD1, syntenin-1 mAb sc-100336, Rab5 mAb sc-46692, mouse Golgin-97 mAb A-21270, ALIX mAb sc-53540, rat HA Ab 3F10 and the mouse mAb anti β-Actin (AC-15) were purchased from Santa Cruz (Dellas, TX, USA), Molecular Probes (Eugene, OR, USA), Roche (Basel, Switzerland), and Sigma-Aldrich (St. Louis, MO, USA), respectively. The LBPA mAb was kindly provided by Jean Gruenberg, Geneva, Switzerland.
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2

Signaling Pathway Antibody Protocol

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The following antibodies were used: Anti-JUNB(ab128878), anti-JUND(ab134067), anti-FOSB(ab184938) and anti-FOSL2(ab124830) were purchased from Abcam (Cambridge, UK); anti-FOS(sc-8047), anti-FOSL1(#sc-183), anti-p-JNK(sc-6254) and anti-β-actin(AC-15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-JUN(#9165), anti-JNK(#9252), anti–p-P38 MAPK(#9211), anti-P38(#9212), anti-p-ERK1/2(#9106), anti-ERK1/2(#9102), anti-ubiquitin(#3936) and anti-p-Rb(#9308) were purchased from Cell Signalling Technology (Danvers, MA, USA); anti-p-JUN (Ser73) (#06–659) was purchased from EMD Millipore (Temecula, CA, USA).
Palbociclib was synthesized by ChemieTek (Indianapolis, IN, USA). SP600125 and MG132 were purchased from Sigma-Aldrich (St Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO). TNFα was purchased from Genzyme (Cambridge, MA, USA) and IL-1β was purchased from PeproTech (Rocky Hill, NJ, USA).
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3

Evaluation of Anti-inflammatory Effects

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Folin-Ciocalteu reagent, aluminum chloride, chlorogenic acid, quercetin, bergapten, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), bovine serum albumin (BSA), protease inhibitors, trypan blue, phosphate buffered saline (PBS), LPS, trichloroacetic acid (TCA), sulphorhodamine B (SRB) were purchased by Sigma-Aldrich S.p.a. (Milan, Italy). ECL System was from Bio-Rad. RAW 264.7 cells were purchased by ATCC, UK (No. TIB-71). The Abs employed were anti-phosphoJak2 (#PA538287), anti-Jak2 (#PA511267), anti-phosphoStat3 (#PA5121259), anti-Stat3(#PA5120138), (all from ThermoFisher Scientific, Waltham, MA, USA), anti-β-actin (AC-15; sc-69879) from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. All other reagents were obtained by VWR International s.r.l. (Milan, Italy).
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IFN-γ and IL1B Biochemical Assay

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Human IFN-γ and interleukin-1B (IL1B) were purchased from Peprotech. The following antibodies were used for biochemical assays: anti-MANF (SAB3500384, Sigma-Aldrich) and anti-β-Actin (AC-15) (sc-69879, Santa Cruz Biotechnology).
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5

Immunoblotting Assay for Fibrillarin Autoantibodies

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The human autoimmune sera with specificity against fibrillarin (O61; 1:3000) was prepared as previously described (Sirri et al., 2002) (link). The phospho-Tyrosine antibody (P-Tyr-100; 1:5000) was from Cell Signaling Technology. The anti-α-tubulin (clone DM1A; 1:1000) and anti-BrdU (clone BU-33; 1:500) antibodies were from Sigma-Aldrich. The anti-SIRT7 (C-3; 1:10,000), anti-β-Actin (AC-15; 1:10,000), anti-ERK 1/2 (C-9; 1:2000) and anti-phospho-ERK (E-4; 1:2000) antibodies were from Santa Cruz Biotechnology. The anti-53BP1 (PA5-54565; 1:3000) and antiphospho-Histone H2AX (Ser139) (CR55T33; 1:3000) antibodies were from Thermo Fisher Scientific. The alkaline phosphatase-conjugated antidigoxigenin (11 093 274 910; 1:20,000) antibody was from Roche. Secondary antibodies coupled to Alexa Fluor 488 1:500), 1:500) and horseradish peroxidase (115-035-003; 1:3000) were from Jackson ImmunoResearch Laboratories, Inc.
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6

Western Blotting and Immunoprecipitation for Huntingtin

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For western blotting mouse monoclonal anti-Htt mAB2166 (Millipore) was used at 1:1000, anti-RACK1 (Gnb2l1) (BD Transduction Laboratories) at 1:2500, anti-Rps6 2217 (Cell Signaling Technology) at 1:500, anti-Myo5a (Cell Signaling Technology) 1:1000, anti-β-tubulin (Covance) 1:1000, anti-β-actin ac15 (Santa Cruz Biotechnology) 1:400, and anti-GFP (Santa Cruz Biotechnology) at 1:500 in TBST. For immunofluorescence, mouse monoclonal anti-FLAG M2 (Sigma) was used at 1:1000, rabbit polyclonal anti-Htt (Enzo Life Sciences) at 1:1000, and anti-Fmr1 (Millipore) mAB2160 at 1:500. For immunoprecipitations 2μg of anti-Htt (Enzo Life Sciences) was used to immunoprecipitate Htt from Neuro-2a cells or 2μg of rabbit IgG (Santa Cruz Biotechnology) as the negative control.
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7

Chromatin Immunoprecipitation Protocol

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Antibodies used were directed against anti-H3 (Abcam, #ab1791), anti-H3.3 (Abcam, #ab176840), anti-H3K9me3 (Abcam, ab8898), anti-H3K36me3 (Abcam, #ab9050), anti-γH2A.X/phospho-histone H2A.X (Ser139) clone JBW301 (Merck Millipore, #05-636), anti-ATRX (Santa Cruz Biotechnologies, #sc15408), anti- KDM4B (Abcam, #ab191434) anti-IDH1 (Sigma Aldrich, SAB4100064), anti-IDH1R132H (Sigma Aldrich, SAB4200548), anti-HP1α (Merck Millipore, #MAB3584), anti-PML (Merck Millipore, #MAB3738), anti-TP53 (Cell Signaling Technologies, #cst-2524), anti-Flag (Sigma, #F1804), anti-TERF1 (Alpha Diagnostics, #TRF12-S) and anti-BrdU (Abcam, #ab6326), Anti-β Actin (AC-15) (Santa Cruz Biotechnology, #sc69879), Goat anti Rabbit IgG, HRP conjugate (Merck Millipore, #AP187P), Donkey anti-Mouse IgG HRP conjugate (Merck Millipore, #AP192P), Donkey anti-Mouse IgG (H + L) Alexa Fluor 594 (Invitrogen, #A-21203), Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, #A-21206).
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8

Antibody detection and immunofluorescence

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Mouse antibodies anti-E7 (1/1000), anti-E6 2E-3F8 (1/1000) and anti-βactin AC-15 (1/10000) were purchased from Santa Cruz, Euromedex and Sigma, respectively. Mouse and rabbit anti-eIF4A3 antibodies were generously provided by Catherine Tomasetto (I.G.B.M.C., Illkirch, Strasbourg). HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from BD Pharmingen. Alexa 488-labeled goat anti-rabbit and Cy3-labeled donkey anti-mouse secondary antibodies used for immunofluorescence were purchased from Jackson ImmunoResearch.
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