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3 protocols using rabbit anti igf1rβ

1

Immunofluorescent Staining: Detailed Protocols

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For immunofluorescent staining, sections were rinsed in PBS and blocked with 5% normal serum/0.1% Triton X-100/PBS at room temperature. Primary antibodies were diluted in blocking solution and incubated 1–2 days at 4°C with gentle agitation. The antibodies utilized were; rabbit anti-Parvalbumin (Swant), chicken anti-EGFP (Aves Labs), rat anti-CTIP2 (Abcam), rabbit anti-SATB2 (Abcam), rabbit anti-CUX1 (Santa Cruz), mouse anti-NEUN (Chemicon), rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology), rabbit anti-IBA1 (Wako), goat anti-IGF1 (R&D Research), rabbit anti-PKCγ (Santa Cruz), rabbit anti-MAP2K1/2(MEK1/2) (Abcam), rabbit anti-P-MAPK1/3(ERK1/2) (Cell Signaling Technology) and rabbit anti-IGF1Rβ (Cell signaling Technology). After rinsing in PBS/T, the secondary antibody was diluted in blocking solution and added overnight at 4°C. Secondary antibodies included Alexa Fluor 488, 546 or 568, and 647 conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat IgG (Invitrogen). For some experiments slides were then incubated in Hoechst or DAPI for nuclear labeling, rinsed, and mounted. Images were collected with a Zeiss LSM 710, 780, or Leica SP5 laser scanning confocal microscope.
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2

Antibody Inventory for Organelle Studies

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Antibodies used in this study were: From Abcam: rabbit anti–CI-MPR (ab124767), rabbit anti-LAMP1 (ab24170), rabbit anti-GLUT1 (ab15309), rabbit anti-VPS35 (ab97545), rabbit anti-VPS26 (ab181352), rabbit anti-VPS29 (ab98929), mouse anti-SNX27 (ab77799), rabbit anti-SNX5 (ab18520), and rabbit anti-INSR (ab131238). From BD: mouse anti-EEA1 (610457), mouse anti-SNX1 (611482), and mouse anti-SNX2 (611308). From AbD Serotec/Bio-Rad Laboratories: sheep anti-TGN46 (AHP500) and mouse anti–CI-MPR (anti-CD222; clone MEM-238). Further antibodies used were: rabbit anti–IGF1R β (9750; Cell Signaling Technology), mouse anti-GAPDH (10494-1-AP; Proteintech), rabbit anti-GGA2 (10356-1-AP; Proteintech), rabbit anti-AP1B1 (16932-1-AP; Proteintech), mouse anti-SNX6 (S6324; Sigma-Aldrich), rat anti-myc (clone 9E1; ChromoTek), and rat anti-GFP (clone 3H9; ChromoTek).
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3

Adenovirus-Mediated Protein Flux Analysis

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Adenovirus encoding CMV promoter-driven GFP:Cre or GFP alone were purchased from Vector Biolabs. Adenovirus encoding mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) reporter was kindly provided by Dr. Qiangrong Liang (23). Recombinant human insulin solution was purchased from Sigma, whereas recombinant human IGF-1 was from PeproTech. Bafilomycin A1 was from Cell Signaling Technology. Rabbit anti-p62 (#23214), rabbit anti-LC3A/B (#12741), rabbit anti-Ulk1 (#8054), rabbit anti-IGF1Rβ (#9750), rabbit anti-GAPDH (#5174) were purchased from Cell Signaling Technology. Rabbit anti-IRβ (#sc-711) was from Santa Cruz. Mouse anti-GFAP (#MAB306) and mouse anti-NeuN (#MAB377B) were purchased from Millipore. Rabbit anti-GFAP (#ab7260) was purchased from Abcam. Rabbit anti-Iba-1 (#019-19741) was purchased from Fujifilm-Wako. HRP-conjugated goat-anti-rabbit IgG secondary antibodies were purchased from GE Healthcare.
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