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5 protocols using anti golgin97

1

Antibodies and Lectins for Cell Analysis

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The following antibodies and lectins were used: anti-myc (mouse, clone 4A6; Millipore; catalog no.: 05-724), anti-BACE1 (mouse, clone 1A11, generous gift from Dr Bart De Strooper (40 (link))), anti-GAPDH (mouse, clone 6C5; Merck Millipore; catalog no.: MAB374), anti-Golgin-97 (rabbit, clone D8P2K, Cell Signaling Technology; catalog no.: 13192S), anticalnexin (rabbit, abcam; catalog no.: ab22595), anti-N-cadherin (rabbit, abcam; catalog no.: ab18203), horseradish peroxidase (HRP)–conjugated antimouse IgG (GE Healthcare; catalog no.: NA931V), HRP-conjugated anti-rabbit IgG (GE Healthcare, Amersham; catalog no.: NA934V), DSA (J-Chemical; catalog no.: J105), SSA letin (J-Chemical; catalog no.: J118), biotinylated RCA (Vector Laboratories; catalog no.: B-1085), Alexa546-conjugated anti-rabbit IgG (Invitrogen; catalog no.: A10040), and Alexa488-conjugated antimouse IgG (Invitrogen; catalog no.: A21202). DSA and SSA were conjugated to HRP using a Peroxidase Labeling Kit-NH2 (Dojindo), according to the manufacturer’s protocol.
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2

Immunofluorescence Staining of Cellular Proteins

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Following antibodies were used: anti-GAPDH (HRP-60004, Proteintech), anti-6∗His (ab18184, Abcam), anti-GFP (11814460001, Roche), anti-Myc (2278S, Cell Signaling Technology), anti-ACBD3 (H00064746-B01P, Abnova), anti-P-PKA-substrate(9621S, Cell Signaling Technology), anti-Golgin97 (13192, Cell Signaling Technology), anti-mCherry (ab167453, Abcam), anti-PKA-Cα (#4782, Cell Signaling Technology), anti-PKA-RIIα (612242, BD), anti-γ-COP (sc-393615, Santa Cruz), anti-GM130 (610822, BD Bioscience), anti-SNAP tag (P9310S, NEB), anti-Clathrin HC (ab21679, Abcam), anti-ARF1 (NB300-505, Novus), anti-β-Actin (3700S, Cell Signaling), and anti-α-Tubulin (3873S, Cell Signaling). Anti-Rabbit Alexa Fluor 488 (A21441), Alexa Fluor 568 (A10042), Alexa Fluor 647 (A21245), and anti-Mouse Alexa Fluor 488 (A21200), Alexa Fluor 568 (A10037), and Alexa Fluor 647 (A21236) for immunofluorescence staining were obtained from ThermoFisher. D/D Solubilizer (635054) was purchased from Clontech. Forskolin (F6886) was purchased from Sigma.
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3

Antibody and Lectin Toolkit for Cell Biology

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The following antibodies and lectins were used: anti-GnT-V (mouse, clone 706824, R&D Systems), anti-α1 sodium potassium ATPase (mouse, clone 464.6, Abcam), anti-LAMP1 (rabbit, Abcam), anti-GAPDH (mouse, clone 6C5, Merck Millipore), anti-GRP78 (BiP) (rabbit, Abcam), anti-GM130 (rabbit, clone D6B1) (Cell Signaling Technology), anti-Golgin97 (rabbit, clone D8P2K, Cell Signaling Technology), anti-Rab5 (rabbit, clone C8B1, Cell Signaling Technology), anti-Rab7 (rabbit, Cell Signaling Technology), anti-Rab11 (rabbit, Cell Signaling Technology), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare), HRP-conjugated anti-rabbit IgG (GE Healthcare), unconjugated-L4-PHA (J-Chemical), biotinylated-SSA (J-Chemical), biotinylated-RCA (Vector Laboratories), FITC-conjugated L4-PHA (J-Oil Mills), Rhodamine-conjugated L4-PHA (Vector Laboratories), Alexa546-conjugated anti-mouse IgG (Invitrogen), Alexa488-conjugated anti-rabbit IgG (Invitrogen). L4-PHA was conjugated to HRP using a Peroxidase Labeling Kit-NH2 (Dojindo) as described previously (22 (link)).
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4

Immunofluorescence Labeling of Cellular Proteins

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Primary antibodies used were anti-FLAG (Sigma-Aldrich, F1804), anti-Myc and anti-HA (Medical & Biological Laboratories Co., M192-3S and M180-3, respectively), anti-LOX (Abcam, ab31238), anti-ATP7A (Abcam, ab13995), and anti–Golgin-97 (Cell Signaling Technology, 13192) antibodies. Secondary antibodies used were horseradish peroxidase–conjugated anti-mouse immunoglobulin G (IgG), anti-rabbit IgG, and anti-chicken IgY (Abcam, ab205719, ab6721, and ab6877, respectively) and Alexa Fluor 488–conjugated anti-mouse IgG and Alexa Fluor 555–conjugated anti-rabbit IgG (Thermo Fisher Scientific, A-11029 and A-31572, respectively).
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5

Quantitative Western Blotting Analysis

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Lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total protein was quantified using REVERT stain (Li-Cor Biosciences). Primary antibodies were used according to manufacturer recommendations: anti-MUC16 [X75] (Novus Biologicals, NB6001468), anti-human cathepsin D (Cell Signaling, #2284), anti-mouse cathepsin D (Novus Biologicals, AF1029), anti-cathepsin B [D1C7Y] (Cell Signaling, #31718), anti-cathepsin L [33/2] (Santa Cruz, sc-32320), anti-LAMP1 [1D4B] (Developmental Studies Hybridoma Bank), anti-VDAC1 (Cell Signaling, #4661), anti-GOLGIN-97 (Cell Signaling, #13192), anti-CALR (Cell Signaling #12238), and anti-CATLASE (Cell Signaling, #14097). Secondary antibodies were used according to manufacturer recommendations.
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