Mouse or human iNeuron fractions in NuPage LDS sample buffer with 4 mM β-mercaptoethanol were run on 4–12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Phosphosolutions 1:1000), Rab4 (Abcam 1:1000), KIF5C (Goldstein 1:500), DIC (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Hip1 (Novus Biological 1:1000), KDEL (Abcam 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), HAP1 (Santa Cruz 1:1000), Rab11 (Abcam 1:1000), Rab5 (Abcam, 1:1000), or Syntaxin17 (Juhasz, 1:500) for 16 h at 4 °C. Blots were then incubated with anti-mouse or anti-rabbit secondary HRP-conjugated antibodies (ThermoFisher 1:1000) and imaged using a BioRad Chemi-doc system with Pierce ECL or diluted Femto substrate (1:5 in TBS). Images from 3 to 5 blots were quantified using ImageLab. Comprehensive list of reagents and antibodies used in this study can be found in Table S1 .
Hrp conjugated antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRP-conjugated antibodies are laboratory reagents used in various immunoassay techniques. They consist of an antibody molecule chemically linked to the enzyme Horseradish Peroxidase (HRP). The HRP enzyme can catalyze a chromogenic or chemiluminescent reaction, allowing for the detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Anti xpress, by Thermo Fisher Scientific (2 mentions)
Anti gemin5, by Novus Biologicals (2 mentions)
Semi dry electrotransfer, by Bio-Rad (2 mentions)
Immunodetection of tubulin, by Merck Group (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Rab11, by Abcam (1 mentions)
Cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lentiviral plko 1 plasmids, by Merck Group (1 mentions)
Recombinant human β ngf, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by PerkinElmer (1 mentions)
Enhanced chemiluminescence kit, by PerkinElmer (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Anti cbp, by Abcam (1 mentions)
Rabbit anti doublecortin dcx, by Abcam (1 mentions)
Rabbit anti actin, by Cell Signaling Technology (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Rack1, by Santa Cruz Biotechnology (1 mentions)
12 protocols using hrp conjugated antibodies
1
Immunoblot Analysis of Neuronal Proteins
2
Plasmid Generation and Antibody Protocols
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Expression plasmids for Flag-KIF1Bβ and mutants were described previously (Li et al. 2016 (link)). Plasmids encoding Flag- KIF1Bβ Q98L and Flag-KIF1Bβ T335P were generated using QuikChange II XL site-directed mutagenesis kit (Stratagene). Primers are listed in Supplemental Table S2 . Lentiviral pLKO.1 plasmids encoding shRNAs for human KIF1Bβ were purchased from Sigma, and lentiviral pGFP-c-shLenti plasmids encoding shRNAs for rat KIF1Bβ were purchased from Origene target sequences (Supplemental Table S2 ). Plasmid encoding TRKA-mCherry (pEZ-M56-TRKA) was purchased from Genecopoeia, Inc.
Antibodies are listed inSupplemental Table S3 . Fluorophore-conjugated secondary antibodies were from Molecular Probes, and HRP-conjugated antibodies were purchased from Thermo Scientific. Recombinant human β-NGF was purchased from Peprotech.
Antibodies are listed in
3
Western Blot Analysis of Cell Signaling
4
Immunodetection of Xpress-tagged Gemin5
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Equal amounts of total protein were resolved on SDS-PAGE and transferred to a 0.2 μm pore PVDF membrane (Bio-Rad) using a semi-dry electrotransfer (Bio-Rad). Xpress-G5845-1508 proteins were immunodetected using anti-Gemin5 (Novus), anti-Xpress (Thermo Fisher Scientific), or anti-CBP (Abcam) antibodies. Immunodetection of tubulin (Merck) was used as the loading control. The appropriate secondary HRP-conjugated antibodies (Thermo Fisher Scientific) were used according to the instructions of the manufacturer.
5
Immunofluorescence Staining of Neural Markers
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The following antibodies were used: mouse/rabbit anti-drebrin, mouse/rabbit anti-βIII Tubulin, rabbit anti-doublecortin (DCX) (Abcam); rabbit anti-actin (Cell Signaling), anti-GFAP (Dako), anti-GFP (Invitrogen), rabbit anti-pS142 drebrin [15 (link)]; mouse anti-PSA-NCAM (Sigma). HRP-conjugated antibodies were from Thermo Scientific. Biotinylated HRP antibodies were from Dako. Alexa Fluor 488- and Texas Red-conjugated secondary antibodies were from Invitrogen. Unless otherwise specified, all chemicals were from Sigma and all cell culture reagents were from Invitrogen.
6
Immunodetection of Ribonucleoprotein Complexes
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Equal amounts of total protein were resolved on SDS–polyacrylamide gels and transferred to a PVDF membrane (0.2 μm pore, Bio-Rad) using a semi-dry electrotransfer (Bio-Rad). Gemin5 was immunodetected using anti-Gemin5 (Novus) antibody or anti-Xpress (Thermo Fisher Scientific). Commercial antibodies were used to detect RPL3 and RACK1 (Santa Cruz). P1 and P2 ribosomal proteins were detected with the monoclonal antibody 3BH5 [63 (link)]. Anti-H3A (abcam) and Histone H2A (Cell Signaling) were a kind gift from Dr. C. Gutierrez and Dr. E. Lecona. Immunodetection of tubulin (Merck) was used as loading control. The appropriate secondary HRP-conjugated antibodies (Thermo Fisher Scientific) were used according to the instructions of the manufacturer.
7
Western Blot Analysis of Protein Expression
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Cells were homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail) on ice for 30 min, centrifuged at 14,000g for 15 min at 4°C, and the supernatants were collected as the cell lysates. Equal amounts of protein from each sample were separated by SDS-PAGE on 12% gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Used primary antibodies are listed in Table 2 . Membranes were incubated with the primary antibodies overnight at 4°C, and then with their corresponding secondary HRP-conjugated antibodies (1:15000, Thermo Fisher Scientific, Temecula, CA, USA). Protein signal was detected using SuperSignal West Pico Chemiluminescent system (Thermo Fisher Scientific, Temecula, CA, USA). Western blots were scanned and densitometric analysis was performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistical analyses were performed using paired t-test. The criterion for significance was set at 5%.
8
Investigating FOXO Signaling in Flies
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mCherry::foxo flies were raised on normal food and 0 to 3-day-old adults were transferred for 14 days on respective diets at 20°C (male to female ratio 1:3; food renewal in regular intervals). Adults were snap-frozen with liquid nitrogen and fly heads removed. Fly heads were homogenized with a pestle in sample buffer (loading buffer, please see https://openwetware.org ) and subsequently heat-inactivated for 10 min. Homogenates were centrifuged and supernatants used for TRIS-SDS-PAGE. Separated proteins were transferred by Tank-Blotting to nitrocellulose membrane. Membranes were blocked for 1 h with 5% BSA in 0.1% Triton X-100/PBS and then incubated with antibodies in blocking solution overnight. Polyclonal antibodies used to probe were Akt-pSer505 (Cell Signaling, 4054S), Akt-pThr308 (Invitrogen, 44-602G), Akt (Invitrogen, MAS14916), mCherry (Invitrogen, PAS-34974), and Tubulin (Cell Signaling, 2144S). After washing, membranes were incubated with HRP conjugated antibodies (Thermo Fischer, 31466) for 1 h and then bands detected with chemiluminescence.
9
Quantitative Protein Expression Analysis
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For Western Blot analysis total cellular protein lysates were separated by SDS-PAGE and blotted on PVDF membranes. After the protein transfer membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) and washed six times in TBS. For protein detection primary antibodies anti-Rho A, anti-ROCK and anti-vinculin (Sigma Aldrich Co. LLc, US) were incubated overnight at 4°C followed by a labeling with a horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark) for 1 h at room temperature. Protein signals were visualized by using SuperSignal West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, USA) for detection of peroxidase activity from HRP-conjugated antibodies (Thermo Fisher Scientific Inc., Rockford, USA. Band intensity was analyzed densitometricly with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad Laboratories, Hercules, USA). Each protein detection was repeated at least three times with individual prepared cell lysates from independent passaged cells.
10
Immunophenotyping of Tumor-Infiltrating Immune Cells
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Sections from tumors were cut into 4 μm in thickness and deparaffinized in xylene for 10 min. The slides were immersed in 3% H2O2 for 20 min to block the endogenous peroxidase and were blocked in goat serum blocking solution for 30 min. After being incubated at 4 °C overnight with primary antibodies, the slides were incubated with secondary HRP-conjugated antibodies (Thermo Fisher Scientific) for 30 min at RT. The primary antibodies were as follows: F4/80 (Cell Signaling Technology, 70076S, 1:200), iNOS (Abcam, ab3523, 1:50), CD206 (proteintech, 18,704–1-AP, 1:200), CD4 (Cell Signaling Technology, 25229S, 1:200), CD8 (Cell Signaling Technology, 98941S, 1:200), Granzyme B (NOVUS, NB100–684, 1:200), and PD-1 (Abcam, ab214421, 1:200). IHC stainings were examined with microscopy.
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