Irdye 680rd donkey anti rabbit

The following antibodies were used in this study: mouse anti-β-actin (Abcam, Cambridge, UK; cat# ab6276), mouse anti-γH2AX-S139 (Millipore, Burlington, MA, USA; cat# 05-636), mouse anti-c-Myc (Santa Cruz, Dallas, TX, USA; cat# sc-42), rabbit anti-cleaved PARP Asp214 (Cell Signaling, Danvers, MA, USA; cat# 9541), rabbit anti-MTH1 (Novus Biologicals, Centennial, CO, USA; cat# NB100-109), rabbit anti-p53 pS15 (Cell Signaling; cat# 9284), mouse anti-p53 (Santa Cruz; cat# sc-126), mouse anti-GAPDH (Abcam; cat# ab8245), rat anti-RPA32 (Cell Signaling; cat# 2208).
The secondary antibodies used were: goat anti-rat Alexa Fluor^® 568 (Life Technologies, Carlsbad, CA, USA; cat# A-11077), goat anti-rat Alexa Fluor^® 647 (Life Technologies; cat# A-21247), IRDye^® 800CW donkey anti-rabbit (LI-COR, Lincoln, NE, USA; cat# 926-32213), IRDye^® 680RD donkey anti-rabbit (LI-COR; cat# 926-68073), IRDye^® 800CW donkey anti-mouse (LI-COR; cat# 926-32212), IRDye^® 680RD donkey anti-mouse (LI-COR; cat# 926-68072).

Primary antibodies that were used in immunocytochemistry (ICC) and live antibody-uptake are as follows: rabbit antibody against synaptotagmin 1 (Syt1) (mouse cortical cultures, Oyster550-labelled, 1:100, #105103C3, Synaptic Systems), VGLUT1 (rat cortical cultures, 1:1000, #135303, Synaptic Systems), pSer133 of CREB (1:650, #9198 L, CST), mouse antibody against Syt1 (rat cortical cultures, Oyster550-labelled, 1:250, #105311C3, Synaptic Systems) and against VGLUT1 (mouse cultures, 1:250 #135311, Synaptic Systems). Primary antibodies that were used in western blotting are: guinea pig antibody against synapsin 1 (Syn1, 1:1000, #106104, Synaptic System), rabbit primary antibodies against pSer553Syn1 site (human and mouse pSer553 corresponds to pSer551 in rat Syn1, 1:1000, #ab32532, Abcam) and against pSer62Syn1 site (1:1000, #PA5-38336, Thermo Fisher). All the fluorescent secondary antibodies used in ICC were purchased from Jackson Immunoresearch and are as follows: anti-rabbit Alexa 488 (1:1000, #711545152), anti-mouse Cy5 (1:1000, #715175150) and anti-rabbit Cy3 (1:500 for mouse cultures, #711165152). For fluorescent detection of WB, IRDye 800CW donkey anti-guinea pig (#926-32411) and IRDye 680RD donkey anti-rabbit (# 926-6807) from Li-COR were used.

MCF-10A cells were harvested and lysed in 2 mL of ice cold sample buffer containing 7 mL of 0.5 Tris-HCl (Sigma-Aldrich), 3 mL glycerol (Sigma-Aldrich), and 1 g of sodium dodecyl sulfate (SDS) (Sigma-Aldrich) mixed with 1.2 mg of bromophenol blue (Sigma-Aldrich). 30 ug of protein lysates with 3 μL β-mercaptoethanol (Sigma-Aldrich) were loaded on a 10% polyacrylamide gel (Sigma-Aldrich), electrophoresed, and then transferred to a nitrocellulose membrane (Fisher). Membranes were incubated overnight with primary antibodies against β-actin (Sigma-Aldrich, A2066), E-cadherin (Cell Signaling Technology, 14472 s), β-tubulin (R&D Systems, MAB1195), or vimentin (Cell Signaling Technology, 5741 s). This was followed by incubation for 2 h with a secondary antibody solution containing Li-Cor TBS blocking solution (Li-Cor) and either IRDye 800CW goat anti-rabbit (Li-Cor, 926–32211) and IRDye 680RD donkey anti-mouse (Li-Cor, 926–68072) or IRDye 800CW goat anti-mouse (Li-Cor, 926–68070) or IRDye 680RD donkey anti-rabbit (Li-Cor, 926–32214) antibodies and then imaging on a Li-Cor Odyssey FC (Li-Cor). Protein abundance was normalized to either β-tubulin or β-actin for quantification of western blot data.

All samples were suspended in SDS sample buffer, treated with 1000 U of Endoglycosidase Hf or Endo H (respectively, for molecular weight proteins ~10-50 kDa or ~50–100 kDa; New England Biolabs, P0703S or P0702S) where indicated, and denatured for 1 h at 37 °C prior to resolution by SDS-PAGE (16% PAGE, 120 V, 120 min). To analyse the translation products, gels were fixed for 5 min (20% (v/v) MeOH, 10% (v/v) AcOH), dried for 2 h at 65 °C and the radiolabelled species visualised using a Typhoon FLA-700 (GE Healthcare) following exposure to a phosphorimaging plate for 24–72 h. Knock-down efficiencies (EMC2, EMC5, Sec61α, SRα) and controls (EMC6, OST, LMNB1, hSnd2, CAML) were determined by quantitative immunoblotting. Following transfer to a PVDF membrane in transfer buffer (0.06 M Tris, 0.60 M glycine, 20% MeOH) at 300 mA for 2.5 h, PVDF membranes were incubated in 1X Casein blocking buffer (10X stock from Sigma-Aldrich, B6429) made up in TBS, incubated with appropriate primary antibodies (1:500 or 1:1000 dilution) and processed for fluorescence-based detection as described by LI-COR Biosciences using appropriate secondary antibodies (IRDye 680RD Donkey anti-Goat, IRDye 680RD Donkey anti-Rabbit, IRDye 800CW Donkey anti-Guinea pig, IRDye 800CW Donkey anti-Mouse) at 1:10,000 dilution. Signals were visualised using an Odyssey CLx Imaging System (LI-COR Biosciences).