Evaluating GPNMB ACD mutants by Western Blotting and immunofluorescence.

Seed HEK293 cells.

A) For Western Blotting, we recommend seeding 2.5 x 10^5 cells per well of a 12-well culture plate.

B) For Immunofluorescence, we recommend seeding 1.2 x 10^5 cells per well of a 24-well culture plate. Seed cells on Poly-D-Lysine coated coverslips.

Note: we routinely culture HEK293T cells in DMEM containing 10% FCS.

Incubate in a tissue culture incubator .

This protocol uses a DNA: Fugene ratio of 1:3. Prepare the transfection complexes as follows:

A	B	C	D
	DNA	Fugene	Serum-free DMEM
12 well	1000 ng	3 ul	100 ul
24 well	500 ng	1.5 ul	50 ul

Preparation of transfection complexes (quantities are per well)

Add the required amount of plasmid DNA to serum-free DMEM. Mix briefly.

Add the required amount of Fugene HD Transfection Reagent.

Vortex and incubate for 0h 10m 0s at Room temperature .

In the meantime, change the medium on the cells to fresh DMEM containing 10 % FCS

Add 50µL per 24-well or 100µL per 12-well drop-wise to the cells and incubate

This section describes the sample preparation and analysis for Western Blotting.

Wash the cells gentle with PBS

Block membranes in 5% milk/TBS-T for 1h 0m 0s at Room temperature .

Incubate the membranes with primary antibodies at 4°C .

A	B	C	D	E
Target	Cat #	Supplier	Raised in	Dilution
GPNMB (N-terminal)	AF2550	R&D Systems	Goat	1:1000
GFP	MA5-15256	ThermoFisher Scientific	Mouse	1:1000
Actin	A1978	Sigma	Mouse	1:5000

Table 1: Primary antibodies for Western Blotting.

Wash the plots in TBS-T for 0h 5m 0s at Room temperature. Repeat this step twice for a total of three washes.

Dilute the secondary antibody in 5% milk/TBS-T and incubate the membranes with secondary antibodies at 4Room temperature for 0h 45m 0s.

A	B	C
Antibody suggestion		Dilution
anti-mouse-Peroxidase	eg, A3682, Sigma	1:10000
anti-goat-Peroxidase	eg, A5420, Sigma	1:10000

Table 2: Secondary antibodies for Western Blotting.

Develop the blots using an appropriate developer. Full-length GPNMB-EGFP is detected as a double band at ~125 kDa. A cleaved C-terminal GPNMB fragment can be detected with the anti-GFP antibody at ~35 kDa.

Immediately add 100µL per well of ice-cold cell lysis buffer and place cells on ice.

Scrape cells with a cell scraper and harvest cell lysate into a 1.5 ml Eppendorf tube.

Incubate the cells On ice for 0h 10m 0s , vortex occasionally

Clear the cell lysate by spinning down at 16200x g,4°C,0h 0m 0s for 0h 15m 0s

Transfer the post-nuclear supernatant to a fresh 1.5 ml Eppendorf tube and store at -20°C .

Prepare the cell lysates for Western Blotting by adding LDS sample buffer and denaturing agent and denature the samples at 80°C for 0h 8m 0s.

Run samples on 4-12 % Bis-Tris SDS-page. (approx. 0h 35m 0s at 160 V const.)

Transfer proteins onto a PVDF membrane using the Turbo transfer system (BioRad) or similar.

Gently wash coverslips with PBS and fix in 4 % PFA/PBS for 0h 15m 0s min.

Gently wash the cells with PBS. Replace the PBS and add the permeabilisation/blocking. Incubate coverslips for a minimum of 0h 20m 0s min at Room temperature .

Wash the coverslips once in PBS, stain with DAPI (or other nuclear stain), and mount onto glass slides.

GPNMB can be observed predominantly at the trans-Golgi network but can also be seen at lysosomal compartments.

In the meantime, place a piece of Parafilm onto your bench and label if required. This will act as a flat surface to stain the coverslips on.

Prepare the antibody staining solution in Blocking and staining buffer. Find a suggestion of antibodies for counterstaining below:

A	B	C	D	E
Target	Cat #	Supplier	Raised in	Dilution
LAMP-1	H4A3	DSHB	Mouse	1:100
TGN46	13573-1-AP	Proteintech	Rabbit	1:100

Table 3: Primary antibodies for immunofluorescence.

Pipette a 45µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards.

Incubate for 1h 0m 0s hr in the dark.

Wash the coverslips three times with PBS.

Prepare a staining solution containing the secondary antibody:

A	B
Antibody suggestion	Dilution
anti-mouse-AF647	1:1000
anti-rabbit-AF586	1:1000

Table 4: Secondary antibodies for Immunofluorescence.

Pipette a 45µL drop of the antibody staining solution onto the Parafilm and invert the coverslip onto the staining solution so that the cells face downwards.

Incubate for 0h 45m 0s min in the dark.