Proteins were extracted with whole-cell lysate buffer containing 20 mM Tris-HCl pH 7.4, 140 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% NP40 supplemented with protease inhibitor cocktail (Complete mini, Roche); proteins were quantified with the Bradford assay. Lysates (50 μg of protein) were mixed with Laemmli loading buffer and incubated at 55°C for 5 minutes before being subjected to electrophoresis in a 4-12% acrylamide precast SDS-PAGE gel (BioRad). The following Abs were used for immunoblotting: rabbit anti-human SPPL2a C-term52 (link), rabbit anti-human SPPL2a epitope 8 (“ENLKAVTTEDREMRK”, residues 196-210 of human SPPL2a, provided by Dr. Bernd Schröder), anti-CD74 (ab97479, Abcam), anti-GAPDH HRP (sc-25778, Santa Cruz) and anti-V5-HRP (R96125, Invitrogen) Abs.
R961 25
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The R961-25 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a core device designed for a specific function within the laboratory setting. The description of its core function is provided in a factual and unbiased manner, without any interpretation or extrapolation on its intended use.
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Lab products found in correlation
Anti v5 hrp, by Thermo Fisher Scientific (3 mentions)
Complete mini, by Roche (2 mentions)
Anti gapdh hrp sc 25778, by Santa Cruz Biotechnology (2 mentions)
Ab97479, by Abcam (2 mentions)
Acrylamide precast sds page gel, by Bio-Rad (2 mentions)
A2066, by Merck Group (2 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Donkey anti goat secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cb1001, by Merck Group (1 mentions)
Ab9483, by Abcam (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Clone 3f10, by Roche (1 mentions)
Herc2, by BD (1 mentions)
Ab49900, by Abcam (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
9 protocols using r961 25
1
SPPL2a Protein Extraction and Immunoblotting
2
Evaluating Small GTPase Activation
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GST-PAK1 PBD was used as an effector
for Rac1, RhoH, and Wrch2/RhoV. HEK293T cells were transfected with
the relevant expression construct and lysed after 40 h in buffer (20
mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5 mM β-glycerophosphate,
1 mM DTT) containing 10 μg of GST-PAK1 PBD. The lysate was then
incubated with glutathione-sepharose 4B beads for 45 min at 4 °C.
The beads were washed with lysis buffer and resuspend in LDS sample
buffer. GTP-bound small GTPases were identified by Western blotting
using antibodies specific for Rac1 (05-389, Merck) or anti-V5 (R961-25,
Invitrogen) for RhoH and Wrch2/RhoV.
GST-Rhotekin 1–89
was used as an effector for RhoA, RhoB, and RhoC. Transfected HEK293T
cells were lysed as above, prior to incubation with 100 μL (10
μg) of GST-Rhotekin-bead suspension for 45 min at 4 °C.
Following incubation, the beads were washed and eluted with LDS sample
buffer. GTP-bound RhoA was identified by Western blotting with anti-RhoA
(26C4, sc-418, Santa Cruz Biotechnology Inc.); GTP-bound RhoB with
anti-RhoB (14326-1-AP, Proteintech); and GTP-bound RhoC with anti
RhoC (D40E4, 3430S, Cell Signaling Technology).
for Rac1, RhoH, and Wrch2/RhoV. HEK293T cells were transfected with
the relevant expression construct and lysed after 40 h in buffer (20
mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5 mM β-glycerophosphate,
1 mM DTT) containing 10 μg of GST-PAK1 PBD. The lysate was then
incubated with glutathione-sepharose 4B beads for 45 min at 4 °C.
The beads were washed with lysis buffer and resuspend in LDS sample
buffer. GTP-bound small GTPases were identified by Western blotting
using antibodies specific for Rac1 (05-389, Merck) or anti-V5 (R961-25,
Invitrogen) for RhoH and Wrch2/RhoV.
GST-Rhotekin 1–89
was used as an effector for RhoA, RhoB, and RhoC. Transfected HEK293T
cells were lysed as above, prior to incubation with 100 μL (10
μg) of GST-Rhotekin-bead suspension for 45 min at 4 °C.
Following incubation, the beads were washed and eluted with LDS sample
buffer. GTP-bound RhoA was identified by Western blotting with anti-RhoA
(26C4, sc-418, Santa Cruz Biotechnology Inc.); GTP-bound RhoB with
anti-RhoB (14326-1-AP, Proteintech); and GTP-bound RhoC with anti
RhoC (D40E4, 3430S, Cell Signaling Technology).
3
RalB Protein Interactions Elucidation
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3 × 106 HEK293T cells were seeded in 10 cm dishes 48 h before the end point, in DMEM supplemented with 10% FBS. For RalB/RLIP76 coimmunoprecipitations cells were transfected with GFP (1 μg), flag-RLIP76 (5 μg), and V5-RalB Q72L (5 μg) or GFP only (11 μg) 24 h before the end point, while for RalB/Sec5 coimmunoprecipitations cells were transfected with GFP (1 μg) and V5-RalB Q72L (5 μg) or GFP only (6 μg) 24 h before the endpoint. Dishes were lysed in 1 ml lysis buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1% Triton, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM β-glycerophosphate, and protease inhibitor cocktail (Sigma-Aldrich). Lysates (1 ml) were centrifuged at 17,000g for 20 min and added to Protein G Dynabeads (30 μl, Invitrogen) preincubated with anti-V5 antibody (1 μg, R960-25, Invitrogen). Peptides, at the concentrations indicated in the results, were added and incubated with rotation for 1 h at 4 °C. Precipitated complexes were washed with lysis buffer (3 × 500 μl). Samples were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon-P). Membranes were probed by immunoblotting with the following primary antibodies: anti-V5-HRP (R961-25, Invitrogen), anti-flag-HRP (A8592, Sigma-Aldrich), and anti-Sec5 (EPR9420, Abcam).
4
Quantifying DUX4 Protein Expression
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HEK293 cells were co-transfected (Lipofectamine 2000) with a CMV.DUX4.V5 expression vector containing the DUX4 3′ UTR and U6.miDUX4s or control U6.miLacZ in a 1:5 molar ratio. Protein was extracted in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) 24 hr later and quantified using the DC Protein Assay (Bio-Rad). Fifteen microgram samples were separated on 12% SDS-PAGE, transferred to nitrocellulose membrane, and incubated with the following antibodies: mouse monoclonal antibody to V5 (horseradish peroxidase [HRP]-coupled) (1:5,000, R961-25; Invitrogen); mouse monoclonal GAPDH antibody (1:1,000, CB1001; Millipore), or goat polyclonal GAPDH (1:500, ab9483; Abcam) overnight at 4°C. GAPDH-probed blots were washed and then incubated with HRP-coupled goat anti-mouse or HRP-coupled donkey anti-goat secondary antibody (1:100,000, 115-035-003 and 705-035-147; Jackson ImmunoResearch) for 1 hr at room temperature. Following washes, blots were developed using Immobilon Western HRP substrate (Millipore) and exposed to film. DUX4.V5 quantification was assessed using ImageJ.
5
Visualizing IRF7 and IKKε Interactions
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RD cells cotransfected with V5-tagged IRF7 and each Flag-tagged IKKε isoform were fixed with 3.7% paraformaldehyde and permeabilized with 0.5% Triton-X100/PBS. After incubation in blocking buffer (1%BSA/PBS), slides were incubated sequentially with primary antibody (anti-Flag, Proteintech 20543-1-AP, and anti-V5, Invitrogen #R961-25) and secondary antibody (Goat anti-Rabbit Alexa 594 and Goat anti-Mouse Alexa 488). Nuclei were counterstained with Hoechst33342 (Sigma 14533).
6
Protein Detection Techniques in Biological Research
7
Western Blot Quantification of LDLR and PCSK9
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Following the incubation times and treatments specific to each experiment, cultured cells were washed and lysed and the lysates cleared, as described above. Thirty to fifty micrograms of protein were separated on 8% Tris glycine SDS-PAGE gels and transferred to a PVDF membrane. Western blotting was performed for human LDLR-V5 and human PCSK9-V5 (anti-V5-HRP, 1:5000; R96125; Invitrogen) and for β-actin (rabbit anti-β-actin, 1:5000; A2066; Sigma). After incubation with the appropriate secondary antibodies, if required, the membranes were revealed using Clarity Western ECL Substrate (Bio-Rad), imaged with a GelDoc XR+ instrument (Bio-Rad) and the bands of interest quantified using ImageLab 5.2.1 software (Bio-Rad).
8
Antibody-based Detection of TK2 Isoforms
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Three rabbit polyclonal antibodies were used to detect the TK2 proteins. For the canonical TK2 protein, one antibody (SAB1306026 [1:1000], Sigma-Aldrich) was used to detect the TK2 N-terminus, and another (NBP1-79,890 [1:2000], Novus Biologicals, Centennial, CO, USA) was used to detect its C-terminus. For the TK2-EXT protein, SAB1306026 was used to detect the N-terminus, and our original antibody (rabbit polyclonal antibody “pAb #A18JP00152” against the peptide RHNKQAGRRDGRPGELHVP [1:100]) was used to detect the C-terminus. Hence, SAB1306026 could detect both canonical TK2 and TK2-EXT, whereas NBP1-79,890 and pAb #A18JP00152 specifically detected canonical TK2 and TK2-EXT, respectively. Other antibodies used were rabbit anti-G3PD antibody (ab9485 [1:5000], Abcam, Cambridge, UK), rabbit anti-β-actin antibody (A2066 [1:5000], Sigma-Aldrich), mouse anti-His antibody (ab18184 [1:2000], Abcam), anti-V5-HRP antibody (R961-25 [1:5000], Invitrogen), mouse anti-calbindin-D28k antibody (C9848 [1:5000], Sigma-Aldrich), mouse anti-COX IV antibody (for western blot, sc-376731 [1:1500], Santa Cruz, CA, USA; for IHC, ab14744 [1:100], Abcam), and mouse anti-COX I antibody (for western blot, ab14705 [1:5000], Abcam; for IHC, the same antibody [1:500]).
9
SPPL2a Protein Extraction and Immunoblotting
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